Engineered microbes and methods for microbial oil overproduction from cellulosic materials

ABSTRACT

The invention relates to engineering microbial cells for utilization of cellulosic materials as a carbon source, including xylose.

RELATED APPLICATIONS

This application is a continuation of U.S. provisional patentapplication Ser. No. 13/923,607, filed Jun. 21, 2013, and claimspriority under 35 U.S.C. § 119(e) to U.S. provisional patent applicationSer. No. 61/663,391, filed Jun. 22, 2012, the entire contents of each ofwhich are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.DE-AR0000059 awarded by the Department of Energy. The Government hascertain rights in the invention.

BACKGROUND

Sustainably produced biofuels are an alternative to fossil fuels and mayhelp to alleviate the depletion of easily accessible fossil fuel stocks,such as cellulosic biomass, while avoiding fossil fuel-associatedpollution and greenhouse gas emission, thus satisfying a rising demandfor affordable energy in a sustainable way. The development of methodsand oil-producing organisms suitable for the efficient conversion ofcarbon sources to lipids is prerequisite for widespread implementationof microbial biofuel production.

SUMMARY OF CERTAIN ASPECTS OF THE INVENTION

Microbial oil production by heterotrophic organisms is a most promisingpath for the cost-effective production of biofuels from renewableresources provided high conversion yields can be achieved. The key tocost-effective microbial oil production from renewable feedstocks is ahigh carbohydrate to oil conversion yield. Additionally, the use ofavailable and abundant cellulosic biomass feedstocks for biofuelproduction is currently limited by the high cost and energy associatedwith processing such sources. Metabolic engineering has emerged as theenabling technology applied to this end and numerous examples exist ofsuccessful pathway engineering that markedly improved the performance ofmicrobial biocatalysts in the synthesis of chemical, pharmaceutical andfuel products.

Prior efforts at engineering microbes for oil production have focused onamplifying presumed rate-controlling steps in the fatty acid synthesispathway, using traditional carbon sources such as glucose. Significantdrawbacks of such approaches include the high cost of a glucose-basedfeedstock, and that increasing carbon flux into fatty acid synthesispathways increases the level of saturated fatty acids in the cell, whichactivate a potent negative feedback loop of fatty acid biosynthesis.

Some aspects of this disclosure provide a strategy for microbeengineering that combines the utilization of nontraditional carbonsources, such as those obtained from cellulosic materials, includingxylose, with amplification of upstream (metabolite-forming pathways,also referred to herein as “push”) and downstream (product-sequesteringpathways, also referred to herein as “pull”) metabolic pathways. Someaspects of this invention provide that a balanced combination ofpush-and-pull modifications in a microbe results in large carbon fluxamplifications into lipid synthesis pathways without significantdepartures of the concentrations of intermediate metabolites from theirhomeostatic physiological levels, thus avoiding feedback inhibition oflipid synthesis.

Some aspects of this disclosure provide engineered microbes, and methodsof use thereof, that can utilize carbon sources from cellulosic biomassthat are not typically or efficiently metabolized for lipid synthesis.In some aspects, such a carbon source in cellulosic biomass is xylose.

According to one aspect of the invention, isolated oleaginous cells areprovided. The cells include a genetic modification that increasesexpression of: a) a xylose reductase (XYL1) gene product and a xylitoldehydrogenase (XYL2) gene product; and/or b) a xylose isomerase (XYLA)gene product. In some embodiments, the cells also include a geneticmodification that increases expression of a xylulokinase (XYL3) geneproduct. In some embodiments, the cells also include a geneticmodification that increases expression of a diacylglycerolacyltransferase (DGA) gene product, an acetyl-coA carboxylase (ACC) geneproduct, a stearoyl-CoA-desaturase (SCD) gene product, and/or anATP-citrate lyase (ACL) gene product.

In some embodiments, the genetic modification includes a nucleic acidconstruct that increases the expression of the gene product, the nucleicacid construct comprising (a) an expression cassette comprising anucleic acid sequence encoding the gene product under the control of asuitable homologous or heterologous promoter, and/or (b) a nucleic acidsequence that modulates the level of expression of the gene product wheninserted into the genome of the cell. In certain embodiments, thepromoter is an inducible or a constitutive promoter.

In some embodiments, the promoter is a TEF promoter. In someembodiments, the expression construct further comprises an intron. Incertain embodiments, the intron is downstream of the transcriptioninitiation site. In some preferred embodiments, the intron is within thenucleic acid sequence encoding the gene product.

In some embodiments, the nucleic acid construct inhibits or disrupts thenatural regulation of a native gene encoding the gene product resultingin overexpression of the native gene. In certain embodiments, inhibitionor disruption of the natural regulation of the native gene is mediatedby deletion, disruption, mutation and/or substitution of a regulatoryregion, or a part of a regulatory region regulating expression of thegene.

In some embodiments, the gene product is a transcript. In otherembodiments, the gene product is a protein.

In some embodiments, the nucleic acid construct is inserted into thegenome of the cell.

In some embodiments, the increased expression of the gene productconfers a beneficial phenotype for the conversion of a carbon source toa fatty acid, fatty acid derivative and/or triacylglycerol (TAG) to thecell. In certain embodiments, the beneficial phenotype is a modifiedfatty acid profile, a modified TAG profile, an increased fatty acidand/or triacylglycerol synthesis rate, an increase conversion yield, anincreased triacylglycerol accumulation in the cell, and/or an increasedtriacylglycerol accumulation in a lipid body of the cell. Increased inthis context means increased relative to cells that do not haveincreased expression of the gene product. In some embodiments, thesynthesis rate, yield or accumulation of a fatty acid or a TAG of thecell is at least 2-fold increased as compared to unmodified cells of thesame cell type. In certain embodiments, the synthesis rate, yield oraccumulation of a fatty acid or a TAG of the cell is at least 5-foldincreased as compared to unmodified cells of the same cell type. In someembodiments, the synthesis rate, yield or accumulation of a fatty acidor a TAG of the cell is at least 10-fold increased as compared tounmodified cells of the same cell type.

In some embodiments, the cell converts a carbon source to a fatty acidor a TAG at a conversion rate within the range of about 0.025 g/g toabout 0.32 g/g (g TAG produced/g Glucose consumed). In some embodiments,the cell converts a carbon source to a fatty acid or a TAG at aconversion rate of at least about 0.11 g/g. In some embodiments, thecell converts a carbon source to a fatty acid or a TAG at a conversionrate of at least about 0.195 g/g. In some embodiments, the cell convertsa carbon source to a fatty acid or a TAG at a conversion rate of atleast about 0.27 g/g.

In some embodiments, the cell comprises a lipid body or vacuole.

In some embodiments, the cell is a bacterial cell, an algal cell, afungal cell, or a yeast cell. In certain embodiments, the cell is anoleaginous yeast cell. In preferred embodiments, the cell is a Y.lipolytica cell.

According to another aspect of the invention, cultures are provided thatinclude the oleaginous cells described herein. In some embodiments, theculture also includes a carbon source. In some embodiments, the carbonsource comprises a fermentable sugar. In certain embodiments, thefermentable sugar is a C5 and/or a C6 sugar. In some embodiments, thecarbon source includes glucose. In some embodiments, the carbon sourceincludes xylose. In certain embodiments, the xylose is at aconcentration of about 8% wt./vol. In some embodiments, the carbonsource includes arabitol.

In some embodiments, the carbon source includes glycerol. In certainembodiments, the glycerol is at a concentration of about 2% wt./vol.

In some embodiments, the culture includes a carbon/nitrogen (C/N) ratioof about 100.

According to another aspect of the invention, methods are provided. Themethods includes contacting a carbon source with an isolated oleaginouscell as described herein and incubating the carbon source contacted withthe cell under conditions suitable for at least partial conversion ofthe carbon source into a fatty acid or a triacylglycerol by the cell.

In some embodiments, the carbon source comprises a fermentable sugar. Incertain embodiments, the fermentable sugar is a C5 and/or a C6 sugar. Insome embodiments, the carbon source includes glucose. In someembodiments, the carbon source includes xylose. In certain embodiments,the xylose is at a concentration of about 8% wt./vol. In someembodiments, the carbon source includes arabitol.

In some embodiments, the carbon source includes glycerol. In certainembodiments, the glycerol is at a concentration of about 2% wt./vol.

In some embodiments, the method includes a carbon/nitrogen (C/N) ratioof about 100.

In some embodiments, the carbon source contacted with the isolatedoleaginous cell is incubated in a reactor. In some embodiments, thecarbon source is contacted with the isolated oleaginous cell andincubated for conversion of the carbon source to a fatty acid or atriacylglycerol in a fed batch process. In other embodiments, the carbonsource is contacted with the isolated oleaginous cell and incubated forconversion of the carbon source to a fatty acid or a triacylglycerol ina continuous process.

In some embodiments, the fatty acid or the triacylglycerol is extractedfrom the carbon source contacted with the isolated oleaginous cell bysolvent extraction. In certain embodiments, the solvent extraction is achloroform methanol extraction. In other embodiments, the solventextraction is a hexane extraction.

In some embodiments, the fatty acid or the triacylglycerol is separatedfrom the carbon source contacted with the isolated oleaginous cell andsubsequently refined by transesterification.

According to another aspect of the invention, methods for increasingproductivity of production of fatty acid or triacylglycerol by anoleaginous cell are provided. The methods include culturing anoleaginous cell as described herein or a culture as described hereinwith at least two types of carbon sources, wherein the first type ofcarbon source contains or is xylose, and wherein the second type ofcarbon source is a carbon source other than xylose. In such methods theproductivity of production of fatty acid or triacylglycerol by anoleaginous cell is improved relative to culturing the cell or theculture without the second type of carbon source.

In some embodiments, the second type of carbon source contains or is aC2 carbon source, a C3 carbon source, a C5 carbon source other thanxylose or a C6 carbon source.

In some embodiments, the methods also include culturing the oleaginouscell or the culture and the at least two types of carbon sources underconditions suitable for at least partial conversion of the carbon sourceinto a fatty acid or a triacylglycerol by the cell or the culture.

In some embodiments, the xylose is at a concentration of about 8%wt./vol.

In some embodiments, the second type of carbon source includes glucose.In some embodiments, the second type of carbon source includes arabitol.In some embodiments, the second type of carbon source includes glycerol.In certain embodiments, the glycerol is at a concentration of about 2%wt./vol. In some embodiments, the second type of carbon source comprisescellulosic material.

In some embodiments, the method comprises a carbon/nitrogen (C/N) ratioof about 100.

The subject matter of this application may involve, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of a single system or article.

Other advantages, features, and uses of the invention will be apparentfrom the detailed description of certain non-limiting embodiments, thedrawings, which are schematic and not intended to be drawn to scale, andthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Diagnosing the functionality of endogenous xyloseutilization genes. FIG. 1A: Diagram of utilization pathways for xylose,xylitol, and D-arabitol. FIG. 1B: Shake flask experiments with controlstrain MTYL038 grown on these substrates demonstrate growth onD-arabitol, poor growth on xylitol, and no growth on xylose.

FIG. 2A: Growth of adapted Y. lipolytica strain MTYL081 on xylose assole carbon source in minimal media shake flask, compared to unadaptedMTYL081 and control strain MTYL038 that underwent the adaptationprotocol. FIG. 2B: Transcriptional comparison of the xylose utilizationpathway of an adapted Y. lipolytica strain and an unadapted strain.psXYL1 and psXYL2 are heterologously expressed from S. stipitis, whileylXYL1, ylXYL2, ylXYL3 are the endogenous putative xylose utilizationpathway.

FIGS. 3A-3C. Cofermentation of xylose with glucose (FIG. 3A), glycerol(FIG. 3B), or D-arabitol (FIG. 3C). Cultures were grown on 20 g/L xyloseand 4 g/L of the secondary substrate.

FIG. 4. 2-L bioreactor fermentation of strain MTYL081 on glycerol andxylose. C/N ratio was adjusted to 100, with 20 g/L of glycerol and 80g/L of xylose. Samples were taken in triplicate.

FIG. 5. Comparison of mRNA levels of genes responsible for energyproduction during xylose cofermentation with a secondary substrate:glucose, glycerol, arabitol. The comparison is between two time pointsduring the cofermentation: when primarily the secondary substrate isbeing consumed vs. when the secondary substrate is depleted and onlyxylose is being consumed. Transcript levels that did not changesignificantly are shown in white boxes. Transcript levels that increasedmore than two-fold after transitioning to xylose utilization are shownin dark gray boxes. Transcript levels that decreased more than two-foldafter transitioning to xylose are shown in light gray boxes. Numbersinside of each box indicate the ratio of transcripts during thexylose-only phase vs. secondary substrate phase. Numbers greater than1.0 signify up-regulation of the gene when transitioning from secondarysubstrate to xylose, while numbers less than 1.0 signify downregulation.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Liquid biofuels are a promising alternative to fossil fuels that canhelp ease concerns about climate change and smoothen supplyuncertainties (1). Biodiesel, jet oil and other oil-derived fuels inparticular are necessary for aviation and heavy vehicle transport. Theyare presently produced exclusively from vegetable oils, which is acostly and unsustainable path (2). An attractive possibility is thenon-photosynthetic conversion of renewable carbohydrate feedstocks tooil (3). For biodiesel, a transition from vegetable oil to microbial oilproduction for the oil feedstock presents numerous additionaladvantages: adaptability to diverse feedstocks, flexibility in landrequirements, efficient process cycle turnover, and ease of scale-up(4). In the search for improved feedstocks, the push towards cellulosicbiofuels is a clear choice. Cellulosic biomass mitigates the need tocompete with food crop production; an estimated 1.3+ billion dry tonsper year of biomass is potentially available in the US alone (Perlack2005). Additionally, cellulosic materials can be more efficiently grownand more stably produced compared to sugar crops. However cellulosicmaterials are not naturally consumable by most biofuel-producingorganisms, and thus cellulose requires pretreatment and hydrolysis tobreak the material down into monomeric sugar. The resulting hydrolysatecan then be used as a sugar rich feedstock. Since hydrolysis oflignocellulosic biomass results in 20-30% carbohydrates in the form ofxylose, utilization of pentose sugars is one of the first steps towardefficiently using cellulosic materials.

Another factor in a cost-effective microbial technology for theconversion of carbohydrates to oils is a high carbohydrate to oilconversion yield. Metabolic engineering has emerged as the enablingtechnology applied to this end and numerous examples exist of successfulpathway engineering that markedly improved the performance of microbialbiocatalysts in the synthesis of chemical, pharmaceutical and fuelproducts. Prior efforts at engineering microbes with high lipidsynthesis have focused on amplifying presumed rate-controlling steps inthe fatty acid synthesis pathway. These efforts, however, have producedmixed results, presumably because modulating fatty acid flux gave riseto the levels of saturated fatty acids, which are potent allostericinhibitors of fatty acid biosynthetic enzymes providing a negativefeedback loop for the fatty acid biosynthesis. Certain aspects of thisdisclosure describe an approach that combines the introduction of xylosemetabolic genes to utilize xylose as a carbon source, with theamplification of upstream, metabolite-forming pathways in the lipidsynthesis pathway, with a similar increase in the flux of downstream,metabolite-consuming pathways. Combining the utilization of xylose as acarbon source with a push-and-pull strategy can achieve large fluxamplifications without significant departures of the concentrations ofintermediate metabolites from their homeostatic physiological levels,while growing the cells on a renewable cellulosic carbohydratefeedstock.

The oleaginous yeast Yarrowia lipolytica is an attractive candidate formicrobial oil production, which has also demonstrated usefulness in awide range of other industrial applications: citric acid production,protein production (e.g., proteases and lipases), and bioremediation.With a fully sequenced genome and a growing body of genetic engineeringtools, engineering of Y. lipolytica can be achieved with relative ease.Y. lipolytica also has been found to be robust in culture, able to growon a variety of substrates, and has been used for lipid production onagro-industrial residues, industrial glycerol, and industrial fats. Ithas excellent lipid accumulation capacity, commonly accumulating up to36% of its dry cell weight (DCW) in lipids.

The metabolic pathways for de novo lipid synthesis in Y. lipolytica arebeginning to be fully mapped out. Glucose entering glycolysis enters themitochondria as pyruvate for use in the TCA cycle; however, excessacetyl-coA is transported from the mitochondria to the cytosol via thecitrate shuttle. Cytosolic acetyl-CoA is then converted into malonyl-CoAby acetyl-CoA carboxylase (ACC) as the first step of fatty acidsynthesis. After fatty acid synthesis, triacylglycerol (TAG) synthesisfollows the Kennedy pathway, which occurs in the endoplasmic reticulum(ER) and lipid bodies. Acyl-CoA is the precursor used for acylation tothe glycerol-3-phosphate backbone to form lysophosphatidic acid (LPA),which is further acylated to form phosphatidic acid (PA). PA is thendephosphorylated to form diacylglycerol (DAG) and then a final acylationoccurs by diacylglycerol acyltransferase (DGA) to produce TAG.

Transport of acetyl-CoA from the mitochondria to the cytosol is carriedout by the ATP-citrate lyase (ACL)-mediated cleavage of citrate via thecitrate shuttle yielding Acetyl-CoA and Oxaloacetate (OAA). Acetyl-CoAcarboxylase (ACC) then catalyzes the first committed step towards lipidbiosynthesis, converting cytosolic acetyl-CoA into malonyl-CoA, which isthe primary precursor for fatty acid elongation. Completed fattyacyl-CoA chains are then transported to the endoplasmic reticulum (ER)or lipid body membranes for the final assembly of triacylglycerol (TAG)via the Kennedy pathway. Over 80% of the storage lipids produced in Y.lipolytica are in the form of TAG. Cytosolic OAA is converted to malateby malic dehydrogenase and transported back into the mitochondria tocomplete the citrate shuttle cycle. Reducing equivalents in the form ofNADPH is provided either by the pentose phosphate pathway (PPP) or bymalic enzyme in the transhydrogenase cycle. In Y. lipolytica, high PPPflux and ineffectual malic enzyme overexpression suggest that the formeris the primary source for NADPH.

Instead of utilizing glucose as a carbon source, the metabolicconversion of xylose to lipids is a favorable alternative for reasonsdescribed herein. Xylose enters the cell and can be catabolized througha redox pathway, whereby xylose reductase (XD or XYL1) converts xyloseto xylitol using NADPH as a reducing equivalent. Xylitol is thenconverted to xylulose through the action of xylitol dehydrogenase (XDHor XYL2) using NAD+ as an electron acceptor. Xylulokinase (XK or XYL3)then phosphorylates xylulose to form xylulose-5-P. Alternatively, thexylose isomerase (XYLA) enzyme bypasses the requirement of reducingequivalents, producing xylulose directly from xylose, which is thenconverted to xylulose-5-P by XYL3. Xylulose-5-P can then enter centralmetabolism through the non-oxidative pathway of the PPP where itultimately produces glyceraldehyde-3-phosphate (G3P) andfructose-6-phosphate (F6P). These two products can then enter the restof central metabolism, going through glycolysis to enter the TCA cycle.Production of lipids occurs normally through the transport ofmitochondrial citrate into the cytosol, where it is cleaved by ATPcitrate lyase into oxaloacetate and cytosolic acetyl-coA. The acetyl-coAcan then enter the fatty acid synthesis pathway through the enzymaticactivity of acetyl-coA carboxylase. Acyl-CoA generated from the fattyacid synthase complex are transferred to a glycerol-3-phosphate backboneand ultimately sequestered within lipid bodies as triacylglycerol (TAG).

Intracellular lipid accumulation can occur via two methods: de novolipid synthesis or ex novo incorporation of exogenous fatty acids andlipids. Lipid accumulation most commonly occurs when nutrient supply isexhausted in the presence of excess carbon. In culture, this statetypically coincides with the onset of the stationary phase. In practice,the most commonly used limiting-nutrient is nitrogen, as it is easilycontrollable in media compositions. Despite these inducible conditions,lipid synthesis pathways are highly regulated in order for the organismto balance cell growth with energy storage. For example, ACC alone isregulated at multiple levels and by multiple factors.

This tight regulation was circumvented in certain cases. By eliminatingperoxisomal oxidation pathways and engineering glycerol metabolism, Y.lipolytica was able to achieve 40%-70% lipids through ex novo lipidaccumulation. Coexpression of Δ6- and Δ12-desaturase genes allowed forsignificant production of γ-linolenic acid (GLA) (20). However,engineering lipid biosynthesis pathways in Y. lipolytica is stillrelatively unexplored and strategies are still being developed foreffective engineering of the lipid production pathways to maximizeoutput.

Some aspects of this disclosure provide engineered microbes for theproduction of biofuel or biofuel precursor. The term “biofuel” refers toa fuel that is derived from a biological source, such as a living cell,microbe, fungus, or plant. The term includes, for example, fuel directlyobtained from a biological source, for example, by conventionalextraction, distillation, or refining methods, and fuel produced byprocessing a biofuel precursor obtained from a biological source, forexample by chemical modification, such as transesterificationprocedures. Examples of biofuels that are directly obtainable arealcohols such as ethanol, propanol, and butanol, fat, and oil. Examplesof biofuels that are obtained by processing of a biofuel precursor(e.g., a lipid), are biodiesel (e.g., produced by transesterification ofa lipid), and green diesel/modified oil fuels (e.g., produced byhydrogenation of an oil). Biodiesel, also referred to as fatty acidmethyl (or ethyl) ester, is one of the economically most importantbiofuels today and can be produced on an industrial scale bytransesterification of lipids, in which sodium hydroxide and methanol(or ethanol) reacts with a lipid, for example, a triacylglycerol, toproduce biodiesel and glycerol.

Feedstocks for industrial-scale production of biodiesel include animalfats, vegetable oils, palm oil, hemp, soy, rapeseed, flax, sunflower,and oleaginous algae. In other approaches, biomass is converted by amicrobe into a biofuel precursor, for example, a lipid, that issubsequently extracted and further processed to yield a biofuel. Theterm “biomass” refers to material produced by growth and/or propagationof a living cell or organism, for example, a microbe. Biomass maycontain cells, microbes and/or intracellular contents, for examplecellular fatty acids and TAGS, as well as extracellular material.Extracellular material includes, but is not limited to, compoundssecreted by a cell, for example, secreted fatty acids or TAGs. Importanttypes of biomass for biofuel production are algal biomass andplant-derived biomass, for example, corn stover and wood fiber. In someembodiments, biomass for biofuel or biofuel precursor production maycomprise plant derived sugars, for example, sugarcane or corn derivedsugars.

Some aspects of this disclosure relate to the engineering anddevelopment of a microbial source of lipids, useful, for example, foreconomically viable, industrial-scale biodiesel production. The term“lipid” refers to fatty acids and their derivatives. Accordingly,examples of lipids include fatty acids (FA, both saturated andunsaturated); glycerides or glycerolipids, also referred to asacylglycerols (such as monoglycerides (monoacylgycerols), diglycerides(diacylglycerols), triglycerides (triacylglycerols, TAGs, or neutralfats); phosphoglycerides (glycerophospholipids); nonglycerides(sphingolipids, sterol lipids, including cholesterol and steroidhormones, prenol lipids including terpenoids, fatty alcohols, waxes, andpolyketides); and complex lipid derivatives (sugar-linked lipids orglycolipids, and protein-linked lipids). Lipids are an essential part ofthe plasma membrane of living cells and microbes. Some cells andmicrobes also produce lipids to store energy, for example in the form oftriacylglycerols in lipid bodies, lipid droplets, or vacuoles.

Some aspects of this invention relate to engineered microbes for biofuelor biofuel precursor production. In some embodiments, the microbesprovided herein are engineered to use 5C sugars as a carbon source, forexample xylose. In some embodiments, the microbes provided herein alsoare engineered to optimize their lipid metabolism for lipid production.The term “lipid metabolism” refers to the molecular processes thatinvolve the creation or degradation of lipids. Fatty acid synthesis,fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAGstorage and TAG degradation are examples of processes that are part ofthe lipid metabolism of a cell. Accordingly, the term “fatty acidmetabolism” refers to all cellular or organismic processes that involvethe synthesis, creation, transformation or degradation of fatty acids.Fatty acid synthesis, fatty acid oxidation, TAG synthesis, and TAGdegradation are examples of processes are part of the fatty acidmetabolism of a cell.

The term “triacylglycerol” (TAG, sometimes also referred to astriglyceride) refers to a molecule comprising a single molecule ofglycerol covalently bound to three fatty acid molecules, aliphaticmonocarboxylic acids, via ester bonds, one on each of the glycerolmolecule's three hydroxyl (OH) groups. Triacylglycerols are highlyconcentrated stores of metabolic energy because of their reduced,anhydrous nature, and are a suitable feedstock for biodiesel production.

Many cells and organisms store metabolic energy in the form of fattyacids and fatty acid derivatives, such as TAGs. Fatty acids and theirderivatives, such as TAGs, provide an ideal form to store metabolicenergy. The energy contained in the C—C bonds can be efficientlyreleased by β-oxidation, a reaction formally equivalent to the reverseof fatty acid biosynthesis, but mediated and regulated by differentenzymes constituting a different molecular pathway. Microbes can derivefatty acids from external supply, endogenous turnover, and de novosynthesis. Some aspects of this invention relate to the identificationof a microbe for biofuel or biofuel precursor production based on themicrobe's ability to synthesize and store fatty acids or fatty acidderivatives, such as TAGs, efficiently from an externally suppliedcarbon source.

Natural fatty acid molecules commonly have an unbranched, aliphaticchain, or tail, of 4 to 28 carbon atoms. Fatty acids are referred to as“saturated”, if all carbon atoms of the aliphatic chain are connectedvia a C—C single bond, or as “unsaturated”, if two or more carbon atomsare connected via a C—C double bond. Unsaturated fatty acids playimportant roles in the regulation of membrane fluidity, cellularactivity, metabolism and nuclear events governing gene transcription.

The spectrum of fatty acids in yeast consists mostly of C16 and C18fatty acids, for example palmitic acid (C16), palmitoleic acid (C16),stearic acid (C18) and oleic acid (C18). Palmitic acid is an unbranched,saturated fatty acid, with an aliphatic chain of 16 carbon atoms (carbonatoms/unsaturated bonds: 16.0). Stearic acid is an unbranched, saturatedfatty acid with an aliphatic chain of 18 carbon atoms (18.0).Palmitoleic acid is a monounsaturated fatty acid with an aliphatic chainof 16 carbon atoms (16.1). Oleic acid is a monounsaturated fatty acidwith an aliphatic chain of 18 carbon atoms (18.1). Minor fatty acidspecies in yeast include C14 and C26 fatty acids, which play essentialfunctions in protein modification or as components of sphingolipids andGPI anchors, respectively.

De novo synthesis of fatty acids utilizes substantial amounts ofmetabolites, acetyl-CoA, ATP and NADPH, and thus competes with othercellular processes that are dependent on these compounds. NADPH isrequired for two reduction steps in the fatty acid elongation cycle,linking fatty acid synthesis to the metabolic state of the cell andresults in fatty acid synthesis being restricted to conditions of highenergy load of the cells, indicated by increased ATP/AMP ratio, elevatedreduction equivalents and elevated acetyl-CoA pool. Almost allsubcellular organelles are involved in fatty acid metabolism, indicatingthat maintenance of fatty acid homeostasis requires regulation atmultiple levels. Lipid synthesis steps that generate metabolites,acetyl-CoA, ATP, or NADPH for lipid biosynthesis are sometimes referredto herein as “push steps” of lipid synthesis. The amplification of aprocess that increases the production of a metabolites, acetyl-CoA, ATP,or NADPH for lipid synthesis in a cell, for example, by overexpressing agene product mediating such a metabolite-producing process, is sometimesreferred to herein as a “push modification.”

Most organisms, including yeast, are able to synthesize fatty acids denovo from a variety of carbon sources. In an initial step, acetyl-CoA iscarboxylated by the addition of CO₂ to malonyl-CoA, by the enzymeacetyl-CoA carboxylase (ACC; encoded by ACC1 and HFA1 in yeast). Biotinis an essential cofactor in this reaction, and is covalently attached tothe ACC apoprotein, by the enzyme biotin:apoprotein ligase (encoded byBPL1/ACC2 in yeast). ACC is a trifunctional enzyme, harboring a biotincarboxyl carrier protein (BCCP) domain, a biotin-carboxylase (BC)domain, and a carboxyl-transferase (CT) domain. In most bacteria, thesedomains are expressed as individual polypeptides and assembled into aheteromeric complex. In contrast, eukaryotic ACC, includingmitochondrial ACC variants (Hfa1 in yeast) harbor these functions on asingle polypeptide. Malonyl-CoA produced by ACC serves as a two carbondonor in a cyclic series of reactions catalyzed by fatty acid synthase,FAS, and elongases.

The immediate product of de novo fatty acid synthesis are saturatedfatty acids. Saturated fatty acids are known to be the precursors ofunsaturated fatty acids in eukaryotes, including yeast. Unsaturatedfatty acids are generally produced by desaturation of C—C single bondsin saturated fatty acids by specialized enzymes, called desaturases. Thecontrol mechanisms that govern the conversion of saturated fatty acidsto unsaturated fatty acids are not well understood. In eukaryotes,unsaturated fatty acids play important roles in the regulation ofmembrane fluidity, cellular activity, metabolism and nuclear events thatgovern gene transcription. Typically, about 80% of yeast fatty acids aremonounsaturated, meaning that they contain one unsaturated bond in theiraliphatic chain.

Fatty acids are potent inhibitors of fatty acid synthesis and thefeedback inhibition of fatty acid synthesis by fatty acids is a majorobstacle in engineering microbes for oil production. Some aspects ofthis disclosure are based on the recognition that while pushmodifications of lipid synthesis are typically unable to override fattyacid-mediated feedback inhibition of lipid synthesis, a combination of apush modification (e.g., ACC1 overexpression) with a pull modification(e.g., DGA1 overexpression), can efficiently bypass the feedbackinhibition, thus fully realizing the increased carbon flux to the lipidsynthesis pathway, for example, in TGAs stored in a lipid body orvacuole of the cell

Engineering the Capacity for 5C Sugar Utilization and Increased LipidSynthesis in Oleaginous Microbes

Some aspects of this disclosure provide strategies for engineeringmicrobes for oil production. In some embodiments, such strategies employgenetic engineering of oleaginous microbes, for example, Y. lipolytica,to utilize five carbon (5C) sugars, such as xylose, as a carbon sourcefor lipid synthesis.

Some aspects of this disclosure are based on the surprising discovery,described herein, that oleaginous microbes, such as Y. lipolytica, whichare unable to metabolize xylose for lipid synthesis, can be engineeredto be able to utilize five carbon (5C) sugars as feedstocks or infeedstocks. Some aspects of this disclosure relate to the engineering ofoleaginous microbes to utilize 5C sugars, such as xylose, through theintroduction of exogenous xylose metabolism genes or the amplificationor modification of endogenous xylose metabolism genes. Some aspects ofthis disclosure relate to the discovery that an oleaginous microbe suchas Y. lipolytica has within its genome a copy of an XYL3 gene thatproduces a functional gene product. Some aspects of this disclosure arerelated to the heterologous overexpression of xylose metabolism genes,such as XYL1 and XYL2, or XYLA, in an oleaginous microbe such as Y.lipolytica, which enables the microbe to utilize xylose as a sole carbonsource in the production TAGs.

Some aspects of this disclosure provide strategies for additionalengineering of 5C-utilizing microbes for oil production. In someembodiments, such strategies employ genetic engineering of oleaginousmicrobes, for example Y. lipolytica, to simultaneously amplify a push-and a pull-step of lipid synthesis. Significant increases of lipidproduction in oleaginous yeast host cells can be achieved using thesestrategies.

According to some aspects of this invention, modifying the lipidmetabolism in a microbe in accordance with methods provided herein, forexample by simultaneously overexpressing a gene product mediating ametabolite-generating (push) step and a gene product mediating aproduct-sequestering (pull) step of lipid synthesis, allows for thegeneration of a microbe optimized for use in biofuel or biofuelprecursor production processes. Some aspects of this invention providestrategies and methods for engineering the fatty acid metabolism in amicrobe by simultaneously amplifying a push step and a pull step oflipid biosynthesis, resulting in increased synthesis rate andaccumulation of fatty acids and fatty acid derivatives in the microbe.

Some aspects of this invention provide methods that include geneticmodifications resulting in the modulation of the expression and/oractivity of gene products regulating the lipid metabolism of microbesfor biofuel or biofuel precursor production. Such genetic modificationsaccording to some aspects of this invention are targeted to increasecarbohydrate to fatty acid and/or TAG conversion in order to optimizethe modified microbe for large-scale production of lipids from a carbonsource, for example, a carbohydrate source such as a 5C sugar, e.g.,xylose. Some modifications provided according to some aspects of thisinvention, for example, overexpression, knockout, knock-down, activationand/or inhibition of specific gene products, may be effected alone or incombination, and/or in combination with other modifications known tothose of skill in the art. The term “modification” refers to bothgenetic manipulation, for example, overexpression, knockout, knock-down,activation and/or inhibition of specific gene products, and non-geneticmanipulation, for example, manipulation of the growth media, substrate,substrate pretreatment, pH, temperature, conversion process, etc.

A modification of gene expression, also referred to herein as amodulation of gene expression, can be a disruption or inhibition of thenatural regulation of expression, an overexpression, an inhibition ofexpression, or a complete abolishment of expression of a given gene. Theinsertion of a heterologous promoter upstream of a native gene sequence,for example the native DGA1 or ACC1 gene sequence, or the deletion ofregulatory sequences within a promoter, for example regulatory sequencesthat mediate the feedback inhibition of the DGA1 or ACC1 gene bysaturated fatty acids, are examples of a disruption or inhibition of thenatural regulation of expression. Strategies for the modulation of geneexpression may include genetic alterations, for example by recombinanttechnologies, such as gene targeting or viral transductions, ornon-genetic alterations, for example environmental alterations known toresult in the up- or down-regulation of gene expression, or transientdelivery of modulators, for example drugs or small RNA molecules to thetarget cells. Methods for genetic and non-genetic alterations ofmicrobes are well known to those of skill in the art, and are described,for example, in J. Sambrook and D. Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition(Jan. 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N.Strathern, Methods in Yeast Genetics: A Cold Spring Harbor LaboratoryCourse Manual, Cold Spring Harbor Laboratory Press (April 2005); John N.Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guideto Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods inEnzymology Series, 194), Academic Press (Mar. 11, 2004); ChristineGuthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular andCell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350),Academic Press; 1st edition (Jul. 2, 2002); Christine Guthrie and GeraldR. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C,Volume 351, Academic Press; 1st edition (Jul. 9, 2002); Gregory N.Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, MetabolicEngineering: Principles and Methodologies, Academic Press; 1 edition(Oct. 16, 1998); and Christina Smolke, The Metabolic Pathway EngineeringHandbook: Fundamentals, CRC Press; 1 edition (Jul. 28, 2009), all ofwhich are incorporated by reference herein.

The term “overexpression”, as used herein, refers to an increased levelof expression of a given gene product in a given cell, cell type or cellstate, as compared to a reference cell, for example, a wild type cell ofthe same cell type or a cell of the same cell type but lacking aspecific modification, for example, a genetic modification. Forced,continuous expression of the DGA1 and/or ACC1 gene in Y. lipolyticacells exhibiting concentrations of saturated fatty acids that wouldinhibit DGA1 or ACC1 gene expression in wild-type cells is an example ofgene overexpression.

Some aspects of this invention provide a method for the manipulation ofthe activity of a xylose reductase (XD or XYL1) gene product in amicrobe, including for biofuel or biofuel precursor production. The XYL1gene encodes a reductase that reduces xylose to xylitol, the initialstep of metabolizing xylose as required for entry into the PPP pathway.XYL1 uses NADPH as a reducing equivalent, generating xylitol and NADP+.Xylitol is then acted upon by XYL2 as described herein. In someembodiments, the manipulation is an overexpression. In some embodiments,the manipulation is effected by contacting a microbe for biofuel orbiofuel precursor production with an expression construct comprising anucleic acid coding for a XYL1 gene product, for example, an XD protein,operably linked to a heterologous promoter, for example, a constitutiveor an inducible promoter. In some embodiments, the nucleic acid codingfor a XYL1 gene product comprises the coding sequence of SEQ ID NO: 1.In some embodiments, the XYL1 is Scheffersomyces stipitis XYL1, forexample, S. stipitis XYL1 comprising the amino acid sequence of SEQ IDNO: 2. In some embodiments, the microbe is Y. lipolytica. In someembodiments, manipulation of the activity of a XYL1 gene product in amicrobe is effected to confer a beneficial phenotype for large-scalecarbohydrate to lipid conversion, using xylose as the carbohydratesource. XYL1 gene and gene product sequences are well known to those ofskill in the art. Exemplary, representative gene and gene productsequences can be found under entry XM_001385144 in the NCBI database(www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYL1 nucleic acid andprotein sequences are provided below. Additional suitable XYL1sequences, including sequences from other species, will be apparent tothose of skill in the art, and the invention is not limited in thisrespect.

Xylose Reductase XYL1 DNA (Scheffersomyces stipitis) XM_001385144(SEQ ID NO: 1) TACAACTATACTACAATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTTCGGCTGTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATCGAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCAAGATTGTTGGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAAGAAGGTTGCTTTATAGAGAGGAAATAAAACCTAATATACATTGATTGTACATTT Xylose ReductaseXYL1 Protein (Scheffersomyces stipitis) XP_001385181 (SEQ ID NO: 2)MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLD INLRFNDPWDWDKIPIFV

Some aspects of this invention provide a method for the manipulation ofthe activity of a xylitol dehydrogenase (XDH or XYL2) gene product in amicrobe for biofuel or biofuel precursor production. As describedherein, this manipulation may be made in combination with manipulationof XYL1. The XYL2 gene encodes a dehydrogenase that dehydrogenatesxylitol to xylulose, the second step of metabolizing xylose as requiredfor entry into the PPP. XYL2 uses NAD+ as an electron acceptor,generating xylulose and NADH. Xylulose is then acted upon by XYL3 asdescribed herein. In some embodiments, the manipulation is anoverexpression. In some embodiments, the manipulation is effected bycontacting a microbe for biofuel or biofuel precursor production with anexpression construct comprising a nucleic acid coding for a XYL2 geneproduct, for example, an XDH protein, operably linked to a heterologouspromoter, for example, a constitutive or an inducible promoter. In someembodiments, the nucleic acid coding for a XYL2 gene product comprisesthe coding sequence of SEQ ID NO: 3. In some embodiments, the XYL2 isScheffersomyces stipitis XYL2, for example, S. stipitis XYL2 comprisingthe amino acid sequence of SEQ ID NO: 4. In some embodiments, themicrobe is Y. lipolytica. In some embodiments, manipulation of theactivity of a XYL2 gene product in a microbe is effected to confer abeneficial phenotype for large-scale carbohydrate to lipid conversion,using xylose as the carbohydrate source. XYL2 gene and gene productsequences are well known to those of skill in the art. Exemplary,representative gene and gene product sequences can be found under entryXM_001386945 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYL2 nucleic acid andprotein sequences are provided below. Additional suitable XYL2sequences, including sequences from other species, will be apparent tothose of skill in the art, and the invention is not limited in thisrespect.

Xylitol dehydrogenase XYL2 DNA (Scheffersomyces stipitis) XM_001386945(SEQ ID NO: 3) CCTCACTTTAGTTTGTTTCAATCACCCCTAATACTCTTCACACAATTAAAATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGTTCGAAACTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGTCCAGGTCAAGAAAACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCATGGTAGAATCGGTAACTTCGTTTTGACCAAGCCAATGGTCTTGGGTCACGAATCCGCCGGTACTGTTGTCCAGGTTGGTAAGGGTGTCACCTCTCTTAAGGTTGGTGACAACGTCGCTATCGAACCAGGTATTCCATCCAGATTCTCCGACGAATACAAGAGCGGTCACTACAACTTGTGTCCTCACATGGCCTTCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCACCAGGTACCTTATGTAAGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCCAGACCACGTCAGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTGGTGTCCACGCCTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGCCGTCTTTGGTGCTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCTAAGGGTGTCATCGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCCAAGGACATTGGTGCTGCTACTCACACCTTCAACTCCAAGACCGGTGGTTCTGAAGAATTGATCAAGGCTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAATGTACTGGTGCTGAACCTTGTATCAAGTTGGGTGTTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGTCGGTAACGCTGCTGGTCCAGTCAGCTTCCCAATCACCGTTTTCGCCATGAAGGAATTGACTTTGTTCGGTTCTTTCAGATACGGATTCAACGACTACAAGACTGCTGTTGGAATCTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGACTTTGAACAATTGATCACCCACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCAGAGCCGGTAAGGGTGCTGTCAAGTGTCTCATTGACGGCCCTGAGTAAGTCAACCGCTTGGCTGGCCCAAAGTGAACCAGAAACGAAAATGATTATCAAATAGCTTTATAGACCTTTATCCAAATTTATGTAAACTAATAG Xylitol DehydrogenaseXYL2 Protein (Scheffersomyces stipitis) XP_001386982 (SEQ ID NO: 4)MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAHGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGAKGVIVVDIFDNKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRA GKGAVKCLIDGPE

Some aspects of this invention provide a method for the manipulation ofthe activity of a xylulokinase (XK or XYL3) gene product in a microbefor biofuel or biofuel precursor production. As described herein, thismanipulation may be made in combination with manipulation of XYL1 andXYL2. The XYL3 gene encodes a kinase that uses ATP as a phosphate donor,phosphorylating xylulose to form xylulose-5-P adnADP, the final step ofmetabolizing xylose as required for entry into the PPP. Xylulose-5-Penters the PPP where it ultimately produces glyceraldehyde-3-phosphate(G3P) and fructose-6-phosphate (F6P). These two products can then enterthe rest of central metabolism, going through glycolysis to enter theTCA cycle. Production of lipids occurs normally through pathwaysdescribed herein. In some embodiments, the manipulation is anoverexpression. In some embodiments, the manipulation is effected bycontacting a microbe for biofuel or biofuel precursor production with anexpression construct comprising a nucleic acid coding for a XYL3 geneproduct, for example, an XK protein, operably linked to a heterologouspromoter, for example, a constitutive or an inducible promoter. In someembodiments, the nucleic acid coding for a XYL3 gene product comprisesthe coding sequence of SEQ ID NO: 5. In some embodiments, the XYL3 is Y.lipolytica XYL2, for example, Y. lipolytica XYL2 comprising the aminoacid sequence of SEQ ID NO: 6. In some embodiments, the microbe is Y.lipolytica. In some embodiments, manipulation of the activity of a XYL3gene product in a microbe is effected to confer a beneficial phenotypefor large-scale carbohydrate to lipid conversion, using xylose as thecarbohydrate source. XYL3 gene and gene product sequences are well knownto those of skill in the art. Exemplary, representative gene and geneproduct sequences can be found under entry XM_505266 in the NCBIdatabase (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYL3 nucleic acid andprotein sequences are provided below. Additional suitable XYL3sequences, including sequences from other species, will be apparent tothose of skill in the art, and the invention is not limited in thisrespect.

Xylulokinase XYL3 DNA (Yarrowia lipolytica) XM_505266 (SEQ ID NO: 5)ATGTATCTCGGACTGGATCTTTCGACTCAACAGCTCAAGGGCATCATTCTGGACACAAAAACGCTGGACACGGTCACACAAGTCCATGTGGACTTTGAGGACGACTTGCCGCAGTTCAACACCGAAAAGGGCGTCTTTCACAGCTCTACAGTGGCCGGAGAAATCAATGCTCCTGTGGCAATGTGGGGGGCAGCTGTGGACTTGCTGATAGAGCGTCTGTCAAAGGAAATAGACCTTTCCACGATCAAGTTTGTGTCGGGCTCGTGCCAGCAACACGGCTCTGTTTATCTCAACAGCAGCTACAAGGAGGGCCTGGGTTCTCTGGACAAACACAAAGACTTGTCTACAGGAGTGTCATCCTTACTGGCGCTCGAAGTCAGCCCCAATTGGCAGGATGCAAGCACGGAGAAGGAGTGTGCGCAGTTTGAGGCTGCAGTCGGCGGTCCCGAGCAGCTGGCTGAGATCACTGGCTCTCGAGCACATACTCGTTTCACCGGGCCCCAGATTCTCAAGGTCAAGGAACGCAACCCCAAGGTATTCAAGGCCACGTCACGGGTCCAGCTCATATCCAACTTTCTAGCATCTCTGTTTGCCGGCAAGGCGTGCCCCTTTGATCTTGCTGACGCCTGTGGAATGAATCTGTGGGACATCCAGAATGGCCAGTGGTGCAAGAAACTCACAGATCTCATCACCGATGACACCCACTCGGTCGAGTCCCTCCTTGGAGACGTGGAAACAGACCCCAAGGCTCTACTGGGCAAAATCTCGCCCTATTTCGTCTCCAAGGGCTTCTCTCCCTCTTGTCAGGTGGCACAGTTCACAGGCGACAACCCAGGCACTATGCTGGCTCTCCCCTTACAGGCCAATGACGTGATTGTGTCTTTGGGAACATCTACGACCGCCCTCGTCGTAACAAACAAGTACATGCCCGACCCCGGATACCATGTGTTCAACCACCCCATGGAGGGATACATGGGCATGCTGTGCTACTGCAACGGAGGTCTAGCACGAGAGAAGATCCGAGACGAGCTTGGAGGCTGGGACGAGTTTAATGAGGCGGCCGAGACCACCAACACAGTGTCTGCTGACGATGTCCATGTTGGCATCTACTTTCCACTACGAGAAATCCTTCCTCGAGCAGGTCCCTTTGAACGACGTTTCATCTACAACAGACAAAGTGAACAGCTTACAGAGATGGCTTCTCCAGAGGACTCACTGGCAACCGAACACAAACCGCAGGCTCAAAATCTCAAGGACACGTGGCCGCCACAAATGGACGCCACTGCCATCATTCAAAGCCAGGCCCTCAGTATCAAAATGAGACTCCAACGCATGATGCATGGCGATATTGGAAAGGTGTATTTTGTGGGAGGCGCCTCGGTCAACACTGCTATCTGCAGCGTAATGTCTGCCATCTTAAAACCAACAAAGGGCGCTTGGAGATGTGGTCTGGAAATGGCAAACGCTTGTGCCATTGGAAGTGCCCATCACGCCTGGCTTTGCGACCCCAACAAGACAGGCCAGGTACAGGTTCACGAAGAAGAGGTCAAATACAAGAATGTGGACACAGACGTGCTACTCAAGGCGTTCAAGCTGGCCGAAAACGCCTGCCTGGAGAAATAA Xylulokinase XYL3 Protein (Yarrowia lipolytica)XP_505266 (SEQ ID NO: 6)MYLGLDLSTQQLKGIILDTKTLDTVTQVHVDFEDDLPQFNTEKGVFHSSTVAGEINAPVAMWGAAVDLLIERLSKEIDLSTIKFVSGSCQQHGSVYLNSSYKEGLGSLDKHKDLSTGVSSLLALEVSPNWQDASTEKECAQFEAAVGGPEQLAEITGSRAHTRFTGPQILKVKERNPKVFKATSRVQLISNFLASLFAGKACPFDLADACGMNLWDIQNGQWCKKLTDLITDDTHSVESLLGDVETDPKALLGKISPYFVSKGFSPSCQVAQFTGDNPGTMLALPLQANDVIVSLGTSTTALVVTNKYMPDPGYHVFNHPMEGYMGMLCYCNGGLAREKIRDELGGWDEFNEAAETTNTVSADDVHVGIYFPLREILPRAGPFERRFIYNRQSEQLTEMASPEDSLATEHKPQAQNLKDTWPPQMDATAIIQSQALSIKMRLQRMMHGDIGKVYFVGGASVNTAICSVMSAILKPTKGAWRCGLEMANACAIGSAHHAWLCDPNKTGQVQVHEEEVKYKNVDTDVLLKAFKLAENACLEK

Some aspects of this invention provide a method for the manipulation ofthe activity of a xylose isomerase (XYLA) gene product in a microbe forbiofuel or biofuel precursor production. The XYLA gene encodes anisomerase that converts xylose directly to xylulose without therequirement of reducing equivalents, effectively eliminating one step asdescribed herein with the redox pathway (XYL1/XYL2). Xylulose may thenbe acted upon by XYL3 to form xylulose-5-P, the final step ofmetabolizing xylose as required for entry into the PPP, as describedherein. In some embodiments, the manipulation is an overexpression. Insome embodiments, the manipulation is effected by contacting a microbefor biofuel or biofuel precursor production with an expression constructcomprising a nucleic acid coding for a XYLA gene product, for example, aXYLA protein, operably linked to a heterologous promoter, for example, aconstitutive or an inducible promoter. In some embodiments, the nucleicacid coding for a XYLA gene product comprises the coding sequence of SEQID NO: 7. In some embodiments, the XYLA is Piromyces sp. E2 XYLA, forexample, Piromyces sp. E2 XYLA comprising the amino acid sequence of SEQID NO: 8. In some embodiments, the microbe is Y. lipolytica. In someembodiments, manipulation of the activity of a XYLA gene product in amicrobe is effected to confer a beneficial phenotype for large-scalecarbohydrate to lipid conversion, using xylose as the carbohydratesource. XYLA gene and gene product sequences are well known to those ofskill in the art. Exemplary, representative gene and gene productsequences can be found under GenBank entries HV445113, FW568191, andHC036431 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYLA nucleic acid andprotein sequences are provided below. Additional suitable XYLAsequences, including sequences from other species, will be apparent tothose of skill in the art, and the invention is not limited in thisrespect.

Piromyces sp E2 Xylose isomerase DNA sequence (SEQ ID NO: 7)ATGGCTAAAGAGTACTTCCCACAGATTCAGAAGATAAAGTTCGAGGGCAAAGATTCTAAAAACCCTTTGGCTTTCCACTACTATGATGCAGAGAAGGAAGTCATGGGAAAGAAAATGAAGGATTGGTTGAGATTTGCTATGGCTTGGTGGCATACTTTGTGTGCTGAAGGTGCAGACCAGTTCGGCGGTGGCACTAAGTCTTTTCCTTGGAATGAGGGTACTGATGCCATTGAAATCGCCAAACAAAAGGTAGACGCTGGTTTTGAGATCATGCAGAAGTTGGGCATCCCTTATTACTGTTTTCACGATGTCGATTTGGTGAGTGAAGGCAATAGTATAGAGGAATACGAGTCTAACTTAAAGGCAGTCGTTGCCTATTTGAAGGAGAAGCAAAAGGAAACTGGTATCAAATTGTTGTGGAGTACTGCTAACGTCTTCGGCCACAAAAGATACATGAACGGTGCTTCTACTAATCCAGACTTTGATGTAGTCGCTAGAGCTATAGTCCAGATTAAGAATGCTATCGACGCCGGAATTGAGTTGGGAGCTGAGAACTATGTTTTTTGGGGAGGTAGGGAAGGCTATATGTCTTTGTTGAATACTGACCAGAAGAGAGAGAAAGAACACATGGCAACAATGTTAACTATGGCAAGAGATTACGCAAGGAGTAAGGGCTTTAAGGGCACTTTTTTGATTGAACCTAAGCCTATGGAACCAACTAAACACCAATATGATGTTGACACTGAAACAGCCATCGGTTTCTTGAAGGCCCACAACTTGGATAAAGATTTTAAGGTAAACATTGAGGTCAATCACGCCACCTTGGCCGGTCACACTTTCGAACATGAATTGGCTTGTGCTGTTGATGCTGGAATGTTGGGTTCTATTGATGCAAATAGAGGCGATTATCAGAATGGTTGGGATACTGATCAATTTCCAATCGACCAATACGAATTGGTTCAAGCCTGGATGGAAATCATAAGAGGTGGTGGCTTTGTAACTGGTGGAACTAACTTCGATGCCAAAACAAGAAGAAACTCCACTGACTTGGAGGATATCATTATTGCTCACGTTTCCGGTATGGATGCAATGGCCAGGGCCTTGGAGAACGCTGCTAAGTTGTTACAAGAATCCCCCTACACTAAGATGAAGAAAGAGAGGTACGCATCATTCGATTCTGGAATCGGCAAGGATTTTGAGGACGGAAAGTTGACTTTAGAGCAGGTTTATGAGTACGGTAAAAAGAATGGCGAGCCTAAACAAACCTCTGGTAAGCAGGAATTGTACGAAGCTATTGTCG CAATGTATCAATAAPiromyces sp E2 Xylose Isomerase Protein Sequence (SEQ ID NO: 8)MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMAWWHTLCAEGADQFGGGTKSFPWNEGTDAIEIAKQKVDAGFEIMQKLGIPYYCFHDVDLVSEGNSIEEYESNLKAVVAYLKEKQKETGIKLLWSTANVFGHKRYMNGASTNPDFDVVARAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLNTDQKREKEHMATMLTMARDYARSKGFKGTFLIEPKPMEPTKHQYDVDTETAIGFLKAHNLDKDFKVNIEVNHATLAGHTFEHELACAVDAGMLGSIDANRGDYQNGWDTDQFPIDQYELVQAWMEIIRGGGFVTGGTNFDAKTRRNSTDLEDIIIAHVSGMDAMARALENAAKLLQESPYTKMKKERYASFDSGIGKDFEDGKLTLEQVYEYGKKNGEPKQTSGKQELYEAIVAMYQ

Some aspects of this invention provide a method for the manipulation ofthe activity of a diacylglycerol acyltransferase 1 (DGA1) gene productin a microbe for biofuel or biofuel precursor production. The DGA1 geneencodes an acyltransferase that catalyzes the terminal step oftriacylglycerol (TAG) formation, acylating diacylglycerol using acyl-CoAas an acyl donor. The result of this acyltransferase reaction aretriacylglycerols, which do not exhibit the same inhibitory feedbackeffect on fatty acid synthesis as fatty acids themselves. TAGs aretypically stored in lipid bodies or vacuoles in lipid producing cells.In some embodiments, the manipulation is an overexpression. In someembodiments, the manipulation is effected by contacting a microbe forbiofuel or biofuel precursor production with an expression constructcomprising a nucleic acid coding for a DGA1 gene product, for example, aDGAT2 protein, operably linked to a heterologous promoter, for example,a constitutive or an inducible promoter. In some embodiments, thenucleic acid coding for a DGA1 gene product comprises the codingsequence of SEQ ID NO: 9. In some embodiments, the DGA1 is Y. lipolyticaDGA1, for example, Y. lipolytica DGA1 comprising the amino acid sequenceof SEQ ID NO: 10. In some embodiments, the microbe is Y. lipolytica. Insome embodiments, manipulation of the activity of a DGA1 gene product ina microbe is effected to confer a beneficial phenotype for large-scalecarbohydrate to lipid conversion, for example increased lipid synthesisrate, increased carbohydrate to lipid conversion efficiency, increasedlipid storage and, increased growth rate, increased tolerance toelevated concentrations of a carbon source or a lipid product. DGA1 geneand gene product sequences are well known to those of skill in the art.Exemplary, representative gene and gene product sequences can be foundunder entry XM_504700 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of DGA1 nucleic acid andprotein sequences are provided below. Additional suitable DGA1sequences, including sequences from other species, will be apparent tothose of skill in the art, and the invention is not limited in thisrespect.

>gi|50554582|ref|XM_504700.1|Yarrowia lipolyticaYALI0E32769p (YALI0E32769g) mRNA, complete cds (SEQ ID NO: 9)ATGACTATCGACTCACAATACTACAAGTCGCGAGACAAAAACGACACGGCACCCAAAATCGCGGGAATCCGATATGCCCCGCTATCGACACCATTACTCAACCGATGTGAGACCTTCTCTCTGGTCTGGCACATTTTCAGCATTCCCACTTTCCTCACAATTTTCATGCTATGCTGCGCAATTCCACTGCTCTGGCCATTTGTGATTGCGTATGTAGTGTACGCTGTTAAAGACGACTCCCCGTCCAACGGAGGAGTGGTCAAGCGATACTCGCCTATTTCAAGAAACTTCTTCATCTGGAAGCTCTTTGGCCGCTACTTCCCCATAACTCTGCACAAGACGGTGGATCTGGAGCCCACGCACACATACTACCCTCTGGACGTCCAGGAGTATCACCTGATTGCTGAGAGATACTGGCCGCAGAACAAGTACCTCCGAGCAATCATCTCCACCATCGAGTACTTTCTGCCCGCCTTCATGAAACGGTCTCTTTCTATCAACGAGCAGGAGCAGCCTGCCGAGCGAGATCCTCTCCTGTCTCCCGTTTCTCCCAGCTCTCCGGGTTCTCAACCTGACAAGTGGATTAACCACGACAGCAGATATAGCCGTGGAGAATCATCTGGCTCCAACGGCCACGCCTCGGGCTCCGAACTTAACGGCAACGGCAACAATGGCACCACTAACCGACGACCTTTGTCGTCCGCCTCTGCTGGCTCCACTGCATCTGATTCCACGCTTCTTAACGGGTCCCTCAACTCCTACGCCAACCAGATCATTGGCGAAAACGACCCACAGCTGTCGCCCACAAAACTCAAGCCCACTGGCAGAAAATACATCTTCGGCTACCACCCCCACGGCATTATCGGCATGGGAGCCTTTGGTGGAATTGCCACCGAGGGAGCTGGATGGTCCAAGCTCTTTCCGGGCATCCCTGTTTCTCTTATGACTCTCACCAACAACTTCCGAGTGCCTCTCTACAGAGAGTACCTCATGAGTCTGGGAGTCGCTTCTGTCTCCAAGAAGTCCTGCAAGGCCCTCCTCAAGCGAAACCAGTCTATCTGCATTGTCGTTGGTGGAGCACAGGAAAGTCTTCTGGCCAGACCCGGTGTCATGGACCTGGTGCTACTCAAGCGAAAGGGTTTTGTTCGACTTGGTATGGAGGTCGGAAATGTCGCCCTTGTTCCCATCATGGCCTTTGGTGAGAACGACCTCTATGACCAGGTTAGCAACGACAAGTCGTCCAAGCTGTACCGATTCCAGCAGTTTGTCAAGAACTTCCTTGGATTCACCCTTCCTTTGATGCATGCCCGAGGCGTCTTCAACTACGATGTCGGTCTTGTCCCCTACAGGCGACCCGTCAACATTGTGGTTGGTTCCCCCATTGACTTGCCTTATCTCCCACACCCCACCGACGAAGAAGTGTCCGAATACCACGACCGATACATCGCCGAGCTGCAGCGAATCTACAACGAGCACAAGGATGAATATTTCATCGATTGGACCGAGGAGGGCAAAGGAGCCCCAGAGTTCCGAATGATTGAGTAA >gi|50554583|ref|XP_504700.1|YALI0E32769p[Yarrowia lipolytica] (SEQ ID NO: 10)MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPTFLTIFMLCCAIPLLWPFVIAYVVYAVKDDSPSNGGVVKRYSPISRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAIISTIEYFLPAFMKRSLSINEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSRYSRGESSGSNGHASGSELNGNGNNGTTNRRPLSSASAGSTASDSTLLNGSLNSYANQIIGENDPQLSPTKLKPTGRKYIFGYHPHGIIGMGAFGGIATEGAGWSKLFPGIPVSLMTLTNNFRVPLYREYLMSLGVASVSKKSCKALLKRNQSICIVVGGAQESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAFGENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMHARGVFNYDVGLVPYRRPVNIVVGSPIDLPYLPHPTDEEVSEYHDRYIAELQRIYNEHKDEYFIDW TEEGKGAPEFRMIE

Some aspects of this invention provide a method for the manipulation ofan acetyl-CoA carboxylase (ACC) gene product in a microbe for biofuel orbiofuel precursor production, for example, in Y. lipolytica. ACC geneproducts mediate the conversion of acetyl-CoA, the main C2-precursor infatty acid synthesis, to malonyl-CoA, which is considered the firstcommitted step in fatty acid synthesis and has been suggested to also bethe rate-limiting step in fatty acid synthesis (see Cao Y, Yang J, XianM, Xu X, Liu W. Increasing unsaturated fatty acid contents inEscherichia coli by coexpression of three different genes. ApplMicrobiol Biotechnol. 2010). In some embodiments, ACC activitymanipulation is ACC overexpression. In some embodiments, themanipulation is effected by contacting a microbe for biofuel or biofuelprecursor production with an expression construct comprising a nucleicacid coding for an ACC gene product, for example, an ACC1 protein,operably linked to a heterologous promoter, for example, a constitutiveor an inducible promoter. In some embodiments, the nucleic acid codingfor an ACC gene product comprises the coding sequence of SEQ ID NO: 11.In some embodiments, the ACC gene product is an ACC1 protein comprisingthe amino acid sequence of SEQ ID NO: 12. In some embodiments, ACCoverexpression in a microbe increases fatty acid synthesis rate and/orconfers a beneficial phenotype for large-scale carbohydrate to biofuelor biofuel precursor conversion, for example increased lipid synthesisrate, increased carbohydrate to lipid conversion efficiency, increasedlipid storage and, increased growth rate, increased tolerance toconcentrations of a substance, e.g. a carbon source, a biofuel orbiofuel precursor, or a toxic substance. ACC gene and gene productsequences are well known to those of skill in the art. Exemplary,representative gene and gene product sequences can be found under theentry for GeneIDs: 855750 and 2909424, or under the entry NC_006069 inthe NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of ACC nucleic acid andprotein sequences are provided below. Additional suitable ACC sequences,including sequences from other species, will be apparent to those ofskill in the art, and the invention is not limited in this respect.

ACC encoding nucleic acid sequence: (SEQ ID NO: 11)ATGCGACTGCAATTGAGGACACTAACACGTCGGTTTTTCAGGTGAGTAAACGACGGTGGCCGTGGCCACGACAGCCGAGGCGTCACGATGGGCCAGACGAGCACATTCTCGCCGCCACAACCTCGCCAGCACAAGAAACTAACCCAGTATGGCTTCAGGATCTTCAACGCCAGATGTGGCTCCCTTGGTGGACCCCAACATTCACAAAGGTCTCGCCTCTCATTTCTTTGGACTCAATTCTGTCCACACAGCCAAGCCCTCAAAAGTCAAGGAGTTTGTGGCTTCTCACGGAGGTCATACAGTTATCAACAAGGTGAGTATTTGACGTTTAGACTGTATAACAGGCGGCCGCAGTGCAACAACGACCAAAAAGGGTCGAAAAAGGGTCGAAAACGGACACAAAAGCTGGAAAACAAGAGTGTAATACATTCTTACACGTCCAATTGTTAGACAAACACGGCTGTTCGGTCCCAAAACCACCAGTATCACCTATTTTCCACTTGTGTCTCGGATCTGATCATAATCTGATCTCAAGATGAAATTTACGCCACCGACATGATATTGTGATTTTCGGATTCTCCAGACCGAGCAGATTCCAGCAATACCACCACTTGCCCACCTTCAGCGGCCTCTCGGCGCGATTCGCCACTTTCCCCAACGAGTGTTACTAACCCAGGTCCTCATCGCTAACAACGGTATTGCCGCAGTAAAGGAGATCCGTTCAGTACGAAAATGGGCCTACGAGACCTTTGGCGACGAGCGAGCAATCTCGTTCACCGTCATGGCCACCCCCGAAGATCTCGCTGCCAACGCCGACTACATTAGAATGGCCGATCAGTACGTCGAGGTGCCCGGAGGAACCAACAACAACAACTACGCCAACGTCGAGCTGATTGTCGACGTGGCTGAGCGATTCGGCGTCGATGCCGTGTGGGCCGGATGGGGCCATGCCAGTGAAAATCCCCTGCTCCCCGAGTCGCTAGCGGCCTCTCCCCGCAAGATTGTCTTCATCGGCCCTCCCGGAGCTGCCATGAGATCTCTGGGAGACAAAATTTCTTCTACCATTGTGGCCCAGCACGCAAAGGTCCCGTGTATCCCGTGGTCTGGAACCGGAGTGGACGAGGTTGTGGTTGACAAGAGCACCAACCTCGTGTCCGTGTCCGAGGAGGTGTACACCAAGGGCTGCACCACCGGTCCCAAGCAGGGTCTGGAGAAGGCTAAGCAGATTGGATTCCCCGTGATGATCAAGGCTTCCGAGGGAGGAGGAGGAAAGGGTATTCGAAAGGTTGAGCGAGAGGAGGACTTCGAGGCTGCTTACCACCAGGTCGAGGGAGAGATCCCCGGCTCGCCCATCTTCATTATGCAGCTTGCAGGCAATGCCCGGCATTTGGAGGTGCAGCTTCTGGCTGATCAGTACGGCAACAATATTTCACTGTTTGGTCGAGATTGTTCGGTTCAGCGACGGCATCAAAAGATTATTGAGGAGGCTCCTGTGACTGTGGCTGGCCAGCAGACCTTCACTGCCATGGAGAAGGCTGCCGTGCGACTCGGTAAGCTTGTCGGATATGTCTCTGCAGGTACCGTTGAATATCTGTATTCCCATGAGGACGACAAGTTCTACTTCTTGGAGCTGAATCCTCGTCTTCAGGTCGAACATCCTACCACCGAGATGGTCACCGGTGTCAACCTGCCCGCTGCCCAGCTTCAGATCGCCATGGGTATCCCCCTCGATCGAATCAAGGACATTCGTCTCTTTTACGGTGTTAACCCTCACACCACCACTCCAATTGATTTCGACTTCTCGGGCGAGGATGCTGATAAGACACAGCGACGTCCCGTCCCCCGAGGTCACACCACTGCTTGCCGAATCACATCCGAGGACCCTGGAGAGGGTTTCAAGCCCTCCGGAGGTACTATGCACGAGCTCAACTTCCGATCCTCGTCCAACGTGTGGGGTTACTTCTCCGTTGGTAACCAGGGAGGTATCCATTCGTTCTCGGATTCGCAGTTTGGTCACATCTTCGCCTTCGGTGAGAACCGAAGTGCGTCTCGAAAGCACATGGTTGTTGCTTTGAAGGAACTATCTATTCGAGGTGACTTCCGAACCACCGTCGAGTACCTCATCAAGCTGCTGGAGACACCGGACTTCGAGGACAACACCATCACCACCGGCTGGCTGGATGAGCTTATCTCCAACAAGCTGACTGCCGAGCGACCCGACTCGTTCCTCGCTGTTGTTTGTGGTGCTGCTACCAAGGCCCATCGAGCTTCCGAGGACTCTATTGCCACCTACATGGCTTCGCTAGAGAAGGGCCAGGTCCCTGCTCGAGACATTCTCAAGACCCTTTTCCCCGTTGACTTCATCTACGAGGGCCAGCGGTACAAGTTCACCGCCACCCGGTCGTCTGAGGACTCTTACACGCTGTTCATCAACGGTTCTCGATGCGACATTGGAGTTAGACCTCTTTCTGACGGTGGTATTCTGTGTCTTGTAGGTGGGAGATCCCACAATGTCTACTGGAAGGAGGAGGTTGGAGCCACGCGACTGTCTGTTGACTCCAAGACCTGCCTTCTCGAGGTGGAGAACGACCCCACTCAGCTTCGATCTCCCTCTCCCGGTAAGCTGGTTAAGTTCCTGGTCGAGAACGGCGACCACGTGCGAGCCAACCAGCCCTATGCCGAGATTGAGGTCATGAAGATGTACATGACTCTCACTGCTCAGGAGGACGGTATTGTCCAGCTGATGAAGCAGCCCGGTTCCACCATCGAGGCTGGCGACATCCTCGGTATCTTGGCCCTTGATGATCCTTCCAAGGTCAAGCATGCCAAGCCCTTTGAGGGCCAGCTTCCCGAGCTTGGACCCCCCACTCTCAGCGGTAACAAGCCTCATCAGCGATACGAGCACTGCCAGAACGTGCTCCATAACATTCTGCTTGGTTTCGATAACCAGGTGGTGATGAAGTCCACTCTTCAGGAGATGGTTGGTCTGCTCCGAAACCCTGAGCTTCCTTATCTCCAGTGGGCTCATCAGGTGTCTTCTCTGCACACCCGAATGAGCGCCAAGCTGGATGCTACTCTTGCTGGTCTCATTGACAAGGCCAAGCAGCGAGGTGGCGAGTTTCCTGCCAAGCAGCTTCTGCGAGCCCTTGAGAAGGAGGCGAGCTCTGGCGAGGTCGATGCGCTCTTCCAGCAAACTCTTGCTCCTCTGTTTGACCTTGCTCGAGAGTACCAGGACGGTCTTGCTATCCACGAGCTTCAGGTTGCTGCAGGCCTTCTGCAGGCCTACTACGACTCTGAGGCCCGGTTCTGCGGACCCAACGTACGTGACGAGGATGTCATTCTCAAGCTTCGAGAGGAGAACCGAGATTCTCTTCGAAAGGTTGTGATGGCCCAGCTGTCTCATTCTCGAGTCGGAGCCAAGAACAACCTTGTGCTGGCCCTTCTCGATGAATACAAGGTGGCCGACCAGGCTGGCACCGACTCTCCTGCCTCCAACGTGCACGTTGCAAAGTACTTGCGACCTGTGCTGCGAAAGATTGTGGAGCTGGAATCTCGAGCTTCTGCCAAGGTATCTCTGAAAGCCCGAGAGATTCTCATCCAGTGCGCTCTGCCCTCTCTAAAGGAGCGAACTGACCAGCTTGAGCACATTCTGCGATCTTCTGTCGTCGAGTCTCGATACGGAGAGGTTGGTCTGGAGCACCGAACTCCCCGAGCCGATATTCTCAAGGAGGTTGTCGACTCCAAGTACATTGTCTTTGATGTGCTTGCCCAGTTCTTTGCCCACGATGATCCCTGGATCGTCCTTGCTGCCCTGGAGCTGTACATCCGACGAGCTTGCAAGGCCTACTCCATCCTGGACATCAACTACCACCAGGACTCGGACCTGCCTCCCGTCATCTCGTGGCGATTTAGACTGCCTACCATGTCGTCTGCTTTGTACAACTCAGTAGTGTCTTCTGGCTCCAAAACCCCCACTTCCCCCTCGGTGTCTCGAGCTGATTCCGTCTCCGACTTTTCGTACACCGTTGAGCGAGACTCTGCTCCCGCTCGAACCGGAGCGATTGTTGCCGTGCCTCATCTGGATGATCTGGAGGATGCTCTGACTCGTGTTCTGGAGAACCTGCCCAAACGGGGCGCTGGTCTTGCCATCTCTGTTGGTGCTAGCAACAAGAGTGCCGCTGCTTCTGCTCGTGACGCTGCTGCTGCTGCCGCTTCATCCGTTGACACTGGCCTGTCCAACATTTGCAACGTTATGATTGGTCGGGTTGATGAGTCTGATGACGACGACACTCTGATTGCCCGAATCTCCCAGGTCATTGAGGACTTTAAGGAGGACTTTGAGGCCTGTTCTCTGCGACGAATCACCTTCTCCTTCGGCAACTCCCGAGGTACTTATCCCAAGTATTTCACGTTCCGAGGCCCCGCATACGAGGAGGACCCCACTATCCGACACATTGAGCCTGCTCTGGCCTTCCAGCTGGAGCTCGCCCGTCTGTCCAACTTCGACATCAAGCCTGTCCACACCGACAACCGAAACATCCACGTGTACGAGGCTACTGGCAAGAACGCTGCTTCCGACAAGCGGTTCTTCACCCGAGGTATCGTACGACCTGGTCGTCTTCGAGAGAACATCCCCACCTCGGAGTATCTCATTTCCGAGGCTGACCGGCTCATGAGCGATATTTTGGACGCTCTAGAGGTGATTGGAACCACCAACTCGGATCTCAACCACATTTTCATCAACTTCTCAGCCGTCTTTGCTCTGAAGCCCGAGGAGGTTGAAGCTGCCTTTGGCGGTTTCCTGGAGCGATTTGGCCGACGTCTGTGGCGACTTCGAGTCACCGGTGCCGAGATCCGAATGATGGTATCCGACCCCGAAACTGGCTCTGCTTTCCCTCTGCGAGCAATGATCAACAACGTCTCTGGTTACGTTGTGCAGTCTGAGCTGTACGCTGAGGCCAAGAACGACAAGGGCCAGTGGATTTTCAAGTCTCTGGGCAAGCCCGGCTCCATGCACATGCGGTCTATCAACACTCCCTACCCCACCAAGGAGTGGCTGCAGCCCAAGCGGTACAAGGCCCATCTGATGGGTACCACCTACTGCTATGACTTCCCCGAGCTGTTCCGACAGTCCATTGAGTCGGACTGGAAGAAGTATGACGGCAAGGCTCCCGACGATCTCATGACTTGCAACGAGCTGATTCTCGATGAGGACTCTGGCGAGCTGCAGGAGGTGAACCGAGAGCCCGGCGCCAACAACGTCGGTATGGTTGCGTGGAAGTTTGAGGCCAAGACCCCCGAGTACCCTCGAGGCCGATCTTTCATCGTGGTGGCCAACGATATCACCTTCCAGATTGGTTCGTTTGGCCCTGCTGAGGACCAGTTCTTCTTCAAGGTGACGGAGCTGGCTCGAAAGCTCGGTATTCCTCGAATCTATCTGTCTGCCAACTCTGGTGCTCGAATCGGCATTGCTGACGAGCTCGTTGGCAAGTACAAGGTTGCGTGGAACGACGAGACTGACCCCTCCAAGGGCTTCAAGTACCTTTACTTCACCCCTGAGTCTCTTGCCACCCTCAAGCCCGACACTGTTGTCACCACTGAGATTGAGGAGGAGGGTCCCAACGGCGTGGAGAAGCGTCATGTGATCGACTACATTGTCGGAGAGAAGGACGGTCTCGGAGTCGAGTGTCTGCGGGGCTCTGGTCTCATTGCAGGCGCCACTTCTCGAGCCTACAAGGATATCTTCACTCTCACTCTTGTCACCTGTCGATCCGTTGGTATCGGTGCTTACCTTGTTCGTCTTGGTCAACGAGCCATCCAGATTGAGGGCCAGCCCATCATTCTCACTGGTGCCCCCGCCATCAACAAGCTGCTTGGTCGAGAGGTCTACTCTTCCAACTTGCAGCTTGGTGGTACTCAGATCATGTACAACAACGGTGTGTCTCATCTGACTGCCCGAGATGATCTCAACGGTGTCCACAAGATCATGCAGTGGCTGTCATACATCCCTGCTTCTCGAGGTCTTCCAGTGCCTGTTCTCCCTCACAAGACCGATGTGTGGGATCGAGACGTGACGTTCCAGCCTGTCCGAGGCGAGCAGTACGATGTTAGATGGCTTATTTCTGGCCGAACTCTCGAGGATGGTGCTTTCGAGTCTGGTCTCTTTGACAAGGACTCTTTCCAGGAGACTCTGTCTGGCTGGGCCAAGGGTGTTGTTGTTGGTCGAGCTCGTCTTGGCGGCATTCCCTTCGGTGTCATTGGTGTCGAGACTGCGACCGTCGACAATACTACCCCTGCCGATCCCGCCAACCCGGACTCTATTGAGATGAGCACCTCTGAAGCCGGCCAGGTTTGGTACCCCAACTCGGCCTTCAAGACCTCTCAGGCCATCAACGACTTCAACCATGGTGAGGCGCTTCCTCTCATGATTCTTGCTAACTGGCGAGGCTTTTCTGGTGGTCAGCGAGACATGTACAATGAGGTTCTCAAGTACGGATCTTTCATTGTTGATGCTCTGGTTGACTACAAGCAGCCCATCATGGTGTACATCCCTCCCACCGGTGAGCTGCGAGGTGGTTCTTGGGTTGTGGTTGACCCCACCATCAACTCGGACATGATGGAGATGTACGCTGACGTCGAGTCTCGAGGTGGTGTGCTGGAGCCCGAGGGAATGGTCGGTATCAAGTACCGACGAGACAAGCTACTGGACACCATGGCTCGTCTGGATCCCGAGTACTCCTCTCTCAAGAAGCAGCTTGAGGAGTCTCCCGATTCTGAGGAGCTCAAGGTCAAGCTCAGCGTGCGAGAGAAGTCTCTCATGCCCATCTACCAGCAGATCTCCGTGCAGTTTGCCGACTTGCATGACCGAGCTGGCCGAATGGAGGCCAAGGGTGTCATTCGTGAGGCTCTTGTGTGGAAGGATGCTCGTCGATTCTTCTTCTGGCGAATCCGACGACGATTAGTCGAGGAGTACCTCATTACCAAGATCAATAGCATTCTGCCCTCTTGCACTCGGCTTGAGTGTCTGGCTCGAATCAAGTCGTGGAAGCCTGCCACTCTTGATCAGGGCTCTGACCGGGGTGTTGCCGAGTGGTTTGACGAGAACTCTGATGCCGTCTCTGCTCGACTCAGCGAGCTCAAGAAGGACGCTTCTGCCCAGTCGTTTGCTTCTCAACTGAGAAAGGACCGACAGGGTACTCTCCAGGGCATGAAGCAGGCTCTCGCTTCTCTTTCTGAGGCTGAGCGGGCTGAGCTGCTCAAGGGGTTGTGA >gi|50548503|ref|XP_501721.1|YALI0C11407p[Yarrowia lipolytica] (SEQ ID NO: 12)MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVASHGGHTVINKVLIANNGIAAVKEIRSVRKWAYETFGDERAISFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFGVDAVWAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKISSTIVAQHAKVPCIPWSGTGVDEVVVDKSTNLVSVSEEVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGIRKVEREEDFEAAYHQVEGEIPGSPIFIMQLAGNARHLEVQLLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTFTAMEKAAVRLGKLVGYVSAGTVEYLYSHEDDKFYFLELNPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPHTTTPIDFDFSGEDADKTQRRPVPRGHTTACRITSEDPGEGFKPSGGTMHELNFRSSSNVWGYFSVGNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSIRGDFRTTVEYLIKLLETPDFEDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRASEDSIATYMASLEKGQVPARDILKTLFPVDFIYEGQRYKFTATRSSEDSYTLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTCLLEVENDPTQLRSPSPGKLVKFLVENGDHVRANQPYAEIEVMKMYMTLTAQEDGIVQLMKQPGSTIEAGDILGILALDDPSKVKHAKPFEGQLPELGPPTLSGNKPHQRYEHCQNVLHNILLGFDNQVVMKSTLQEMVGLLRNPELPYLQWAHQVSSLHTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSGEVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLLQAYYDSEARFCGPNVRDEDVILKLREENRDSLRKVVMAQLSHSRVGAKNNLVLALLDEYKVADQAGTDSPASNVHVAKYLRPVLRKIVELESRASAKVSLKAREILIQCALPSLKERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVFDVLAQFFAHDDPWIVLAALELYIRRACKAYSILDINYHQDSDLPPVISWRFRLPTMSSALYNSVVSSGSKTPTSPSVSRADSVSDFSYTVERDSAPARTGAIVAVPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASSVDTGLSNICNVMIGRVDESDDDDTLIARISQVIEDFKEDFEACSLRRITFSFGNSRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQLELARLSNFDIKPVHTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADRLMSDILDALEVIGTTNSDLNHIFINFSAVFALKPEEVEAAFGGFLERFGRRLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVSGYVVQSELYAEAKNDKGQWIFKSLGKPGSMHMRSINTPYPTKEWLQPKRYKAHLMGTTYCYDFPELFRQSIESDWKKYDGKAPDDLMTCNELILDEDSGELQEVNREPGANNVGMVAWKFEAKTPEYPRGRSFIVVANDITFQIGSFGPAEDQFFFKVTELARKLGIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLATLKPDTVVTTEIEEEGPNGVEKRHVIDYIVGEKDGLGVECLRGSGLIAGATSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINKLLGREVYSSNLQLGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGRTLEDGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDSIEMSTSEAGQVWYPNSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRDMYNEVLKYGSFIVDALVDYKQPIMVYIPPTGELRGGSWVVVDPTINSDMMEMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARLDPEYSSLKKQLEESPDSEELKVKLSVREKSLMPIYQQISVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRIRRRLVEEYLITKINSILPSCTRLECLARIKSWKPATLDQGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDRQGTLQGMKQA LASLSEAERAELLKGL.

Some aspects of this invention provide a method for the manipulation ofthe activity of a stearoyl-CoA-desaturase (SCD) in a microbe for biofuelor biofuel precursor production. SCD is a Δ9 desaturase that inserts adouble bond between C9 and C10 of stearic acid coupled to CoA, a keystep in the generation of desaturated fatty acids and their derivatives,as described in more detail elsewhere herein. In some embodiments, themanipulation is an overexpression. In some embodiments, the manipulationis effected by contacting a microbe for biofuel or biofuel precursorproduction with an expression construct comprising a nucleic acid codingfor a SCD gene product, for example, a SCD protein, operably linked to aheterologous promoter, for example, a constitutive or an induciblepromoter. In some embodiments, the nucleic acid coding for an SCD geneproduct comprises the coding sequence of SEQ ID NO: 13. In someembodiments, the SCD is Y. lipolytica SCD, for example, Y. lipolyticaSCD comprising the amino acid sequence of SEQ ID NO: 14. In someembodiments, the microbe is Y. lipolytica. In some embodiments,manipulation of the activity of a SCD in a microbe is effected to confera beneficial phenotype for large-scale carbohydrate to lipid conversion,for example increased lipid synthesis rate, increased carbohydrate tolipid conversion efficiency, increased lipid storage and, increasedgrowth rate, increased tolerance to elevated concentrations of a carbonsource or a lipid product. Stearoyl-CoA Desaturase gene and gene productsequences are well known to those of skill in the art. Exemplary,representative gene and gene product sequences can be found under theentry for GeneID: 852825 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of SCD nucleic acid andprotein sequences are provided below. Additional suitable SCD sequences,including sequences from other species, will be apparent to those ofskill in the art, and the invention is not limited in this respect.

>gi|50548052|ref|XM_501496.1| Yarrowia lipolyticaYALI0C05951p (YALI0C05951g) mRNA, complete cds (SEQ ID NO: 13)ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGAGATGTCAACTACAAGGTCAAGTACACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGGGCCGCCACATTTCCGAGCAGCCCTTCACCTGGGCCAACTGGCACCAGCACATCAACTGGCTCAACTTCATTCTGGTGATTGCGCTGCCTCTGTCGTCCTTTGCTGCCGCTCCCTTCGTCTCCTTCAACTGGAAGACCGCCGCGTTTGCTGTCGGCTATTACATGTGCACCGGTCTCGGTATCACCGCCGGCTACCACCGAATGTGGGCCCATCGAGCCTACAAGGCCGCTCTGCCCGTTCGAATCATCCTTGCTCTGTTTGGAGGAGGAGCTGTCGAGGGCTCCATCCGATGGTGGGCCTCGTCTCACCGAGTCCACCACCGATGGACCGACTCCAACAAGGACCCTTACGACGCCCGAAAGGGATTCTGGTTCTCCCACTTTGGCTGGATGCTGCTTGTGCCCAACCCCAAGAACAAGGGCCGAACTGACATTTCTGACCTCAACAACGACTGGGTTGTCCGACTCCAGCACAAGTACTACGTTTACGTTCTCGTCTTCATGGCCATTGTTCTGCCCACCCTCGTCTGTGGCTTTGGCTGGGGCGACTGGAAGGGAGGTCTTGTCTACGCCGGTATCATGCGATACACCTTTGTGCAGCAGGTGACTTTCTGTGTCAACTCCCTTGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTCCCCGAGACCACGCTCTTACCGCCCTGGTCACCTTTGGAGAGGGCTACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGCCCTCATCTGGTACCAGTACGACCCCACCAAGTGGCTCATCTGGACCCTCAAGCAGGTTGGTCTCGCCTGGGACCTCCAGACCTTCTCCCAGAACGCCATCGAGCAGGGTCTCGTGCAGCAGCGACAGAAGAAGCTGGACAAGTGGCGAAACAACCTCAACTGGGGTATCCCCATTGAGCAGCTGCCTGTCATTGAGTTTGAGGAGTTCCAAGAGCAGGCCAAGACCCGAGATCTGGTTCTCATTTCTGGCATTGTCCACGACGTGTCTGCCTTTGTCGAGCACCACCCTGGTGGAAAGGCCCTCATTATGAGCGCCGTCGGCAAGGACGGTACCGCTGTCTTCAACGGAGGTGTCTACCGACACTCCAACGCTGGCCACAACCTGCTTGCCACCATGCGAGTTTCGGTCATTCGAGGCGGCATGGAGGTTGAGGTGTGGAAGACTGCCCAGAACGAAAAGAAGGACCAGAACATTGTCTCCGATGAGAGTGGAAACCGAATCCACCGAGCTGGTCTCCAGGCCACCCGGGTCGAGAACCCCGGTATGTCTGGCATGGCTGCTTAG >gi|50548053|ref|XP_501496.1|YALI0C05951p[Yarrowia lipolytica] (SEQ ID NO: 14)MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKMSQGAYDDKGRHISEQPFTWANWHQHINWLNFILVIALPLSSFAAAPFVSFNWKTAAFAVGYYMCTGLGITAGYHRMWAHRAYKAALPVRIILALFGGGAVEGSIRWWASSHRVHHRWTDSNKDPYDARKGFWFSHFGWMLLVPNPKNKGRTDISDLNNDWVVRLQHKYYVYVLVFMAIVLPTLVCGFGWGDWKGGLVYAGIMRYTFVQQVTFCVNSLAHWIGEQPFDDRRTPRDHALTALVTFGEGYHNFHHEFPSDYRNALIWYQYDPTKWLIWTLKQVGLAWDLQTFSQNAIEQGLVQQRQKKLDKWRNNLNWGIPIEQLPVIEFEEFQEQAKTRDLVLISGIVHDVSAFVEHHPGGKALIMSAVGKDGTAVFNGGVYRHSNAGHNLLATMRVSVIRGGMEVEVWKTAQNEKKDQNIVSDESGNRIHRAGLQATRVENPGMSGMAA

Some aspects of this invention provide a method for the manipulation ofthe activity of an ATP-citrate lyase (ACL) in a microbe for biofuel orbiofuel precursor production. ACL provides cytosolic acetyl-CoA bycleaving citrate which is shuttled out of the mitochondria as a productof the TCA cycle. Cleaving citrate into oxaloacetate and acetyl-CoA, ACLgene products provide an acetyl-CoA substrate for ACC, which thenmediates the conversion of acetyl-CoA, the main C2-precursor in fattyacid synthesis, to malonyl-CoA, which is considered the first committedstep in fatty acid synthesis, as described in more detail elsewhereherein. In some embodiments, an ACL gene product is a protein composedof two subunits encoded by separate genes. In some embodiments, an ACLgene product is composed of two subunits encoded by the same gene. Insome embodiments, the manipulation is an overexpression. In someembodiments, the manipulation is effected by contacting a microbe forbiofuel or biofuel precursor production with an expression constructcomprising a nucleic acid coding for an ACL gene product, for example,an ACL protein, operably linked to a heterologous promoter, for example,a constitutive or an inducible promoter. In some embodiments, thenucleic acid coding for an ACL gene product comprises the codingsequences of SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiments, theACL is Y. lipolytica ACL, for example, Y. lipolytica ACL comprising theamino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 18. In someembodiments, the microbe is Y. lipolytica. In some embodiments,manipulation of the activity of a ACL in a microbe is effected to confera beneficial phenotype for large-scale carbohydrate to lipid conversion,for example increased lipid synthesis rate, increased carbohydrate tolipid conversion efficiency, increased lipid storage and, increasedgrowth rate, increased tolerance to elevated concentrations of a carbonsource or a lipid product. ATP-citrate lyase gene and gene productsequences are well known to those of skill in the art. Exemplary,representative gene and gene product sequences can be found under theentry for GeneID: 2912101 and 2910381 in the NCBI database(www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of ACL nucleic acid andprotein sequences are provided below. Additional suitable ACL sequences,including sequences from other species, will be apparent to those ofskill in the art, and the invention is not limited in this respect.

ATP Citrate Lyase (Yarrowia lipolytica) subunit 1, ACL1 DNA YALI0E34793gXM_504787 (SEQ ID NO: 15)ATGTCTGCCAACGAGAACATCTCCCGATTCGACGCCCCTGTGGGCAAGGAGCACCCCGCCTACGAGCTCTTCCATAACCACACACGATCTTTCGTCTATGGTCTCCAGCCTCGAGCCTGCCAGGGTATGCTGGACTTCGACTTCATCTGTAAGCGAGAGAACCCCTCCGTGGCCGGTGTCATCTATCCCTTCGGCGGCCAGTTCGTCACCAAGATGTACTGGGGCACCAAGGAGACTCTTCTCCCTGTCTACCAGCAGGTCGAGAAGGCCGCTGCCAAGCACCCCGAGGTCGATGTCGTGGTCAACTTTGCCTCCTCTCGATCCGTCTACTCCTCTACCATGGAGCTGCTCGAGTACCCCCAGTTCCGAACCATCGCCATTATTGCCGAGGGTGTCCCCGAGCGACGAGCCCGAGAGATCCTCCACAAGGCCCAGAAGAAGGGTGTGACCATCATTGGTCCCGCTACCGTCGGAGGTATCAAGCCCGGTTGCTTCAAGGTTGGAAACACCGGAGGTATGATGGACAACATTGTCGCCTCCAAGCTCTACCGACCCGGCTCCGTTGCCTACGTCTCCAAGTCCGGAGGAATGTCCAACGAGCTGAACAACATTATCTCTCACACCACCGACGGTGTCTACGAGGGTATTGCTATTGGTGGTGACCGATACCCTGGTACTACCTTCATTGACCATATCCTGCGATACGAGGCCGACCCCAAGTGTAAGATCATCGTCCTCCTTGGTGAGGTTGGTGGTGTTGAGGAGTACCGAGTCATCGAGGCTGTTAAGAACGGCCAGATCAAGAAGCCCATCGTCGCTTGGGCCATTGGTACTTGTGCCTCCATGTTCAAGACTGAGGTTCAGTTCGGCCACGCCGGCTCCATGGCCAACTCCGACCTGGAGACTGCCAAGGCTAAGAACGCCGCCATGAAGTCTGCTGGCTTCTACGTCCCCGATACCTTCGAGGACATGCCCGAGGTCCTTGCCGAGCTCTACGAGAAGATGGTCGCCAAGGGCGAGCTGTCTCGAATCTCTGAGCCTGAGGTCCCCAAGATCCCCATTGACTACTCTTGGGCCCAGGAGCTTGGTCTTATCCGAAAGCCCGCTGCTTTCATCTCCACTATTTCCGATGACCGAGGCCAGGAGCTTCTGTACGCTGGCATGCCCATTTCCGAGGTTTTCAAGGAGGACATTGGTATCGGCGGTGTCATGTCTCTGCTGTGGTTCCGACGACGACTCCCCGACTACGCCTCCAAGTTTCTTGAGATGGTTCTCATGCTTACTGCTGACCACGGTCCCGCCGTATCCGGTGCCATGAACACCATTATCACCACCCGAGCTGGTAAGGATCTCATTTCTTCCCTGGTTGCTGGTCTCCTGACCATTGGTACCCGATTCGGAGGTGCTCTTGACGGTGCTGCCACCGAGTTCACCACTGCCTACGACAAGGGTCTGTCCCCCCGACAGTTCGTTGATACCATGCGAAAGCAGAACAAGCTGATTCCTGGTATTGGCCATCGAGTCAAGTCTCGAAACAACCCCGATTTCCGAGTCGAGCTTGTCAAGGACTTTGTTAAGAAGAACTTCCCCTCCACCCAGCTGCTCGACTACGCCCTTGCTGTCGAGGAGGTCACCACCTCCAAGAAGGACAACCTGATTCTGAACGTTGACGGTGCTATTGCTGTTTCTTTTGTCGATCTCATGCGATCTTGCGGTGCCTTTACTGTGGAGGAGACTGAGGACTACCTCAAGAACGGTGTTCTCAACGGTCTGTTCGTTCTCGGTCGATCCATTGGTCTCATTGCCCACCATCTCGATCAGAAGCGACTCAAGACCGGTCTGTACCGACATCCTTGGGACGATATCACCTACCTGGTTGGCCAGGAGGCTATCCAGAAGAAGCGAGTCGAGATCAGCGCCGGCGACGTTTCCAAGGCCAAGACTCGATCA TAGATP Citrate Lyase (Yarrowia lipolytica) subunit 1, ACL1 ProteinYALI0E34793p XP_504787 (SEQ ID NO: 16)MSANENISRFDAPVGKEHPAYELFHNHTRSFVYGLQPRACQGMLDFDFICKRENPSVAGVIYPFGGQFVTKMYWGTKETLLPVYQQVEKAAAKHPEVDVVVNFASSRSVYSSTMELLEYPQFRTIAIIAEGVPERRAREILHKAQKKGVTIIGPATVGGIKPGCFKVGNTGGMMDNIVASKLYRPGSVAYVSKSGGMSNELNNIISHTTDGVYEGIAIGGDRYPGTTFIDHILRYEADPKCKIIVLLGEVGGVEEYRVIEAVKNGQIKKPIVAWAIGTCASMFKTEVQFGHAGSMANSDLETAKAKNAAMKSAGFYVPDTFEDMPEVLAELYEKMVAKGELSRISEPEVPKIPIDYSWAQELGLIRKPAAFISTISDDRGQELLYAGMPISEVFKEDIGIGGVMSLLWFRRRLPDYASKFLEMVLMLTADHGPAVSGAMNTIITTRAGKDLISSLVAGLLTIGTRFGGALDGAATEFTTAYDKGLSPRQFVDTMRKQNKLIPGIGHRVKSRNNPDFRVELVKDFVKKNFPSTQLLDYALAVEEVTTSKKDNLILNVDGAIAVSFVDLMRSCGAFTVEETEDYLKNGVLNGLFVLGRSIGLIAHHLDQKRLKTGLYRHPWDDITYLVGQEAIQKKRVEISAGDVSKAKTRSATP Citrate lyase (Yarrowia lipolytica) subunit 2, ACL2 DNA YALI0D24431gXM_503231 (SEQ ID NO: 17)ATGTCAGCGAAATCCATTCACGAGGCCGACGGCAAGGCCCTGCTCGCACACTTTCTGTCCAAGGCGCCCGTGTGGGCCGAGCAGCAGCCCATCAACACGTTTGAAATGGGCACACCCAAGCTGGCGTCTCTGACGTTCGAGGACGGCGTGGCCCCCGAGCAGATCTTCGCCGCCGCTGAAAAGACCTACCCCTGGCTGCTGGAGTCCGGCGCCAAGTTTGTGGCCAAGCCCGACCAGCTCATCAAGCGACGAGGCAAGGCCGGCCTGCTGGTACTCAACAAGTCGTGGGAGGAGTGCAAGCCCTGGATCGCCGAGCGGGCCGCCAAGCCCATCAACGTGGAGGGCATTGACGGAGTGCTGCGAACGTTCCTGGTCGAGCCCTTTGTGCCCCACGACCAGAAGCACGAGTACTACATCAACATCCACTCCGTGCGAGAGGGCGACTGGATCCTCTTCTACCACGAGGGAGGAGTCGACGTCGGCGACGTGGACGCCAAGGCCGCCAAGATCCTCATCCCCGTTGACATTGAGAACGAGTACCCCTCCAACGCCACGCTCACCAAGGAGCTGCTGGCACACGTGCCCGAGGACCAGCACCAGACCCTGCTCGACTTCATCAACCGGCTCTACGCCGTCTACGTCGATCTGCAGTTTACGTATCTGGAGATCAACCCCCTGGTCGTGATCCCCACCGCCCAGGGCGTCGAGGTCCACTACCTGGATCTTGCCGGCAAGCTCGACCAGACCGCAGAGTTTGAGTGCGGCCCCAAGTGGGCTGCTGCGCGGTCCCCCGCCGCTCTGGGCCAGGTCGTCACCATTGACGCCGGCTCCACCAAGGTGTCCATCGACGCCGGCCCCGCCATGGTCTTCCCCGCTCCTTTCGGTCGAGAGCTGTCCAAGGAGGAGGCGTACATTGCGGAGCTCGATTCCAAGACCGGAGCTTCTCTGAAGCTGACTGTTCTCAATGCCAAGGGCCGAATCTGGACCCTTGTGGCTGGTGGAGGAGCCTCCGTCGTCTACGCCGACGCCATTGCGTCTGCCGGCTTTGCTGACGAGCTCGCCAACTACGGCGAGTACTCTGGCGCTCCCAACGAGACCCAGACCTACGAGTACGCCAAAACCGTACTGGATCTCATGACCCGGGGCGACGCTCACCCCGAGGGCAAGGTACTGTTCATTGGCGGAGGAATCGCCAACTTCACCCAGGTTGGATCCACCTTCAAGGGCATCATCCGGGCCTTCCGGGACTACCAGTCTTCTCTGCACAACCACAAGGTGAAGATTTACGTGCGACGAGGCGGTCCCAACTGGCAGGAGGGTCTGCGGTTGATCAAGTCGGCTGGCGACGAGCTGAATCTGCCCATGGAGATTTACGGCCCCGACATGCACGTGTCGGGTATTGTTCCTTTGGCTCTGCTTGGAAAGCGGCCCAAGAATGTCAAGCCTTTTGGCACCGGACCTTCTACTGAGGCTTCCACTCCTCTCGGAGTTTAAATP Citrate lyase (Yarrowia lipolytica) subunit 2, ACL2 ProteinYALI0D24431p XP_503231 (SEQ ID NO: 18)MSAKSIHEADGKALLAHFLSKAPVWAEQQPINTFEMGTPKLASLTFEDGVAPEQIFAAAEKTYPWLLESGAKFVAKPDQLIKRRGKAGLLVLNKSWEECKPWIAERAAKPINVEGIDGVLRTFLVEPFVPHDQKHEYYINIHSVREGDWILFYHEGGVDVGDVDAKAAKILIPVDIENEYPSNATLTKELLAHVPEDQHQTLLDFINRLYAVYVDLQFTYLEINPLVVIPTAQGVEVHYLDLAGKLDQTAEFECGPKWAAARSPAALGQVVTIDAGSTKVSIDAGPAMVFPAPFGRELSKEEAYIAELDSKTGASLKLTVLNAKGRIWTLVAGGGASVVYADAIASAGFADELANYGEYSGAPNETQTYEYAKTVLDLMTRGDAHPEGKVLFIGGGIANFTQVGSTFKGIIRAFRDYQSSLHNHKVKIYVRRGGPNWQEGLRLIKSAGDELNLPMEIYGPDMHVSGIVPLALLGKRPKNVKPFGTGPSTEASTPLGV

Some aspects of this invention provide oleaginous microbes for oilproduction comprising any of the modifications described herein, forexample, in combination with modification of XYL1/XYL2 (and optionallyXYL3) or XYLA: a DGA1 modification as described herein, an ACC1modification as described herein, and/or an SCD modification asdescribed herein. In some embodiments, a modified oleaginous microbe isprovided that comprises a push modification as described herein and apull modification as described herein. In some embodiments, the pushmodification comprises overexpression of an ACC1 gene product. In someembodiments, the pull modification comprises overexpression of a DGA1and/or an SCD gene product.

Some aspects of this invention provide nucleic acids coding for a geneproduct conferring a required and/or desired phenotype for biofuel orbiofuel precursor production to a microbe, for example, Y. lipolytica.In some embodiments, the nucleic acid encodes an XYL1 gene product, forexample, an XYL1 protein. In some embodiments, the nucleic acid encodesan XYL2 gene product, for example, an XYL2 protein. In some embodiments,the nucleic acid encodes an XYL3 gene product, for example, an XYL3protein. In some embodiments, the nucleic acid encodes an XYLA geneproduct, for example, an XYLA protein. In some embodiments, the nucleicacid is a nucleic acid derived from Y. lipolytica. In some embodiments,the nucleic acid encodes a DGA1 gene product, for example, a DGA1protein. In some embodiments, the nucleic acid encodes an ACC1 geneproduct, for example, an ACC1 protein. In some embodiments, the nucleicacid encodes a desaturase, for example a Δ9 desaturase. In someembodiments, the nucleic acid encodes Y. lipolytica Δ9 desaturase (SCD).In some embodiments, a nucleic acid is provided that encodes acombination of gene products, for example in multiple cistrons,comprising a gene product the overexpression of which represents a pushmodification of lipid biosynthesis (e.g., an ACC1 gene product), and agene product the overexpression of which represents a pull modificationof lipid biosynthesis (e.g., a DGA1 and/or SCD gene product).

The term “nucleic acid” refers to a molecule comprising multiple linkednucleotides. “Nucleic acid” and “nucleic acid molecule” are usedinterchangeably and refer to oligoribonucleotides as well asoligodeoxyribonucleotides. The terms also include polynucleosides (i.e.,a polynucleotide minus a phosphate) and any other organic basecontaining nucleic acid. The organic bases include adenine, uracil,guanine, thymine, cytosine and inosine. The nucleic acids may be singleor double stranded. The nucleic acid may be naturally or non-naturallyoccurring. Nucleic acids can be obtained from natural sources, or can besynthesized using a nucleic acid synthesizer (i.e., synthetic).Isolation of nucleic acids are routinely performed in the art andsuitable methods can be found in standard molecular biology textbooks.(See, for example, Maniatis' Handbook of Molecular Biology.) The nucleicacid may be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA,cDNA, rRNA, miRNA, PNA or LNA, or a combination thereof, as describedherein. Non-naturally occurring nucleic acids such as bacterialartificial chromosomes (BACs) and yeast artificial chromosomes (YACs)can also be used in accordance with some aspects of this invention.

Some aspects of this invention relate to the use of nucleic acidderivatives. The use of certain nucleic acid derivatives may increasethe stability of the nucleic acids of the invention by preventing theirdigestion, particularly when they are exposed to biological samples thatmay contain nucleases. As used herein, a nucleic acid derivative is anon-naturally occurring nucleic acid or a unit thereof. Nucleic acidderivatives may contain non-naturally occurring elements such asnon-naturally occurring nucleotides and non-naturally occurring backbonelinkages. Nucleic acid derivatives according to some aspects of thisinvention may contain backbone modifications such as but not limited tophosphorothioate linkages, phosphodiester modified nucleic acids,combinations of phosphodiester and phosphorothioate nucleic acid,methylphosphonate, alkylphosphonates, phosphate esters,alkylphosphonothioates, phosphoramidates, carbamates, carbonates,phosphate triesters, acetamidates, carboxymethyl esters,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof. The backbone composition of the nucleic acids may behomogeneous or heterogeneous.

Nucleic acid derivatives according to some aspects of this invention maycontain substitutions or modifications in the sugars and/or bases. Forexample, some nucleic acid derivatives may include nucleic acids havingbackbone sugars which are covalently attached to low molecular weightorganic groups other than a hydroxyl group at the 3′ position and otherthan a phosphate group at the 5′ position (e.g., an 2′-O-alkylatedribose group). Nucleic acid derivatives may include non-ribose sugarssuch as arabinose. Nucleic acid derivatives may contain substitutedpurines and pyrimidines such as C-5 propyne modified bases,5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, 2-thiouracil and pseudoisocytosine.

In some embodiments, a nucleic acid may comprise a peptide nucleic acid(PNA), a locked nucleic acid (LNA), DNA, RNA, or a co-nucleic acids ofthe above such as DNA-LNA co-nucleic acid.

As used herein the term “isolated nucleic acid molecule” refers to anucleic acid that is not in its natural environment, for example anucleic acid that has been (i) extracted and/or purified from a cell ormicrobe, for example, a bacteria or yeast, by methods known in the art,for example, by alkaline lysis of the host cell and subsequentpurification of the nucleic acid, for example, by a silica adsorptionprocedure; (ii) amplified in vitro, for example, by polymerase chainreaction (PCR); (iii) recombinantly produced by cloning, for example, anucleic acid cloned into an expression vector; (iv) fragmented and sizeseparated, for example, by enzymatic digest in vitro or by shearing andsubsequent gel separation; or (v) synthesized by, for example, chemicalsynthesis. In some embodiments, the term “isolated nucleic acidmolecule” refers to (vi) an nucleic acid that is chemically markedlydifferent from any naturally occurring nucleic acid. In someembodiments, an isolated nucleic acid can readily be manipulated byrecombinant DNA techniques well known in the art. Accordingly, a nucleicacid cloned into a vector, or a nucleic acid delivered to a host celland integrated into the host genome is considered isolated but a nucleicacid in its native state in its natural host, for example, in the genomeof the host, is not. An isolated nucleic acid may be substantiallypurified, but need not be. For example, a nucleic acid that is isolatedwithin a cloning or expression vector is not pure in that it maycomprise only a small percentage of the material in the cell in which itresides. Such a nucleic acid is isolated, however, as the term is usedherein.

Some aspects of this invention relate to nucleic acids encoding a geneproduct conferring a required or desirable phenotype to a microbe forbiofuel or biofuel precursor production which are linked to a promoteror other transcription activating element. In some embodiments, thenucleic acid encoding the gene product and linked to a promoter iscomprised in an expression vector or expression construct. As usedherein, the terms “expression vector” or “expression construct” refer toa nucleic acid construct, generated recombinantly or synthetically, witha series of specified nucleic acid elements that permit transcription ofa particular nucleic acid in a host microbe, for example, an oleaginousyeast. In some embodiments, the expression vector may be part of aplasmid, virus, or nucleic acid fragment. In some embodiments, theexpression vector includes the coding nucleic acid to be transcribedoperably linked to a promoter. A promoter is a nucleic acid element thatfacilitates transcription of a nucleic acid to be transcribed. Apromoter is typically located on the same strand and upstream (or 5′) ofthe nucleic acid sequence the transcription of which it controls. Insome embodiments, the expression vector includes the coding nucleic acidto be transcribed operably linked to a heterologous promoter. Aheterologous promoter is a promoter not naturally operably linked to agiven nucleic acid sequence. For example, the DGA1 gene in Y. lipolyticais naturally operably linked to the Y. lipolytica DGA1 gene promoter.Any promoter other than the wildtype Y. lipolytica DGA1 gene promoteroperably linked to the DGA1 gene, or parts thereof, for example in anexpression construct, would, therefore, be a heterologous promoter inthis context. For example, a TEF1 promoter linked to a nucleic acidencoding a DGA1 gene product is a heterologous promoter in the DGA1context.

In some embodiments, the expression vector includes a coding nucleicacid, for example, a nucleic acid encoding a XYL1 and XYL2 (andoptionally XYL3) gene product, or a XYLA gene product, and optionally aDGA1, ACC1, and/or SCD gene product, operably linked to a constitutivepromoter. The term “constitutive promoter” refers to a promoter thatallows for continual transcription of its associated gene. In someembodiments, the expression vector includes a coding nucleic acid, forexample, a nucleic acid encoding a XYL1 and XYL2 (and optionally XYL3)gene product, or a XYLA gene product, and optionally a DGA1, ACC1,and/or SCD gene product, operably linked to an inducible promoter. Theterm “inducible promoter”, interchangeably used herein with the term“conditional promoter”, refers to a promoter that allows fortranscription of its associated gene only in the presence or absence ofbiotic or abiotic factors. Drug-inducible promoters, for exampletetracycline/doxycycline inducible promoters, tamoxifen-induciblepromoters, as well as promoters that depend on a recombination event inorder to be active, for example the cre-mediated recombination of loxPsites, are examples of inducible promoters that are well known in theart.

Some aspects of this disclosure relate to the surprising discovery thatoverexpression of a given gene product from a heterologous promoter inoleaginous microbes can be significantly enhanced by including an intronin the respective expression construct. Some aspects of this disclosureprovide an intron-enhanced constitutive promoter for gene overexpressionin oleaginous microbes and expression constructs and vectors comprisingthis intron-enhanced promoter. In some embodiments, an intron-enhancedTEF promoter is provided, that comprises a TEF promoter sequence, atranscription start site, an intronic sequence downstream of thetranscription start site, and a coding nucleic acid sequence, forexample, a nucleic acid sequence encoding a XYL1 and XYL2 (andoptionally XYL3) gene product, or a XYLA gene product, and optionally aDGA1, ACC1 and/or SCD gene product. In some embodiments, the intron ispositioned downstream of the translation start site, yet within the openreading frame of the gene sequence, e.g., after the start codon, butbefore the termination site of the nucleic acid sequence encoding thegene product. In some embodiments, the intron is positioned immediatelydownstream of the translation start site, e.g., an ATG start codon, yetupstream of the remainder of the coding sequence. For illustrationpurposes, a non-limiting, exemplary structure of an intron-enhancedexpression construct is provided as follows:

5′-TEF promoter-transcription start site-intron-XYL1 coding sequence-3′.Another non-limiting, exemplary structure of an intron-enhancedexpression construct is provided as follows:

5′-TEF promoter-transcription start site-start codon-intron-XYL1 codingsequence-stop codon-3′. Expression constructs for XYL2, XYL3, XYLA,DGA1, ACC1 and SCD gene products would have the XYL1 coding sequencesubstituted by an XYL2, XYL3, XYLA, DGA1, ACC or SCD coding sequence,respectively.

Suitable TEF promoter sequences as well as suitable intron sequenceswill be apparent to those of skill in the art. Some intron-less TEFpromoter sequences are disclosed, for example, in U.S. Pat. No.6,265,185. Some exemplary, representative sequences are provided below.However, it will be understood that the invention is not limited in thisrespect.

Exemplary TEF Promoter Sequence:

(SEQ ID NO: 19) agagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatca ttcaaa

Exemplary Intron Sequence:

(SEQ ID NO: 20) gtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcag

Exemplary TEF promoter-intron sequence comprising a start codon (ATG)between the promoter and the intron sequences:

(SEQ ID NO: 21) agagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttecttctgagtataagaatca ttcaaa ATGgtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcag

Methods to deliver expression vectors or expression constructs intomicrobes, for example, into yeast cells, are well known to those ofskill in the art. Nucleic acids, including expression vectors, can bedelivered to prokaryotic and eukaryotic microbes by various methods wellknown to those of skill in the relevant biological arts. Methods for thedelivery of nucleic acids to a microbe in accordance to some aspects ofthis invention, include, but are not limited to, different chemical,electrochemical and biological approaches, for example, heat shocktransformation, electroporation, transfection, for exampleliposome-mediated transfection, DEAE-Dextran-mediated transfection orcalcium phosphate transfection. In some embodiments, a nucleic acidconstruct, for example an expression construct comprising a combinationof XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, and/or SCD encoding nucleic acidsequences, is introduced into the host microbe using a vehicle, orvector, for transferring genetic material. Vectors for transferringgenetic material to microbes are well known to those of skill in the artand include, for example, plasmids, artificial chromosomes, and viralvectors. Methods for the construction of nucleic acid constructs,including expression constructs comprising constitutive or inducibleheterologous promoters, knockout and knockdown constructs, as well asmethods and vectors for the delivery of a nucleic acid or nucleic acidconstruct to a microbe are well known to those of skill in the art, andare described, for example, in J. Sambrook and D. Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rdedition (Jan. 15, 2001); David C. Amberg, Daniel J. Burke; and JeffreyN. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor LaboratoryCourse Manual, Cold Spring Harbor Laboratory Press (April 2005); John N.Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guideto Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods inEnzymology Series, 194), Academic Press (Mar. 11, 2004); ChristineGuthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular andCell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350),Academic Press; 1st edition (Jul. 2, 2002); Christine Guthrie and GeraldR. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C,Volume 351, Academic Press; 1st edition (Jul. 9, 2002); Gregory N.Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, MetabolicEngineering: Principles and Methodologies, Academic Press; 1 edition(Oct. 16, 1998); and Christina Smolke, The Metabolic Pathway EngineeringHandbook: Fundamentals, CRC Press; 1 edition (Jul. 28, 2009), all ofwhich are incorporated by reference herein.

In some embodiments, the native promoter of a gene encoding a geneproduct conferring a required or desirable phenotype to a microbe, forexample, the native XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, or SCD promoter,is modified in the microbe to alter the regulation of itstranscriptional activity. In some embodiment, the modified promoterexhibits an increased transcriptional activity as compared to itsunmodified counterpart. The term “modified promoter”, as used herein,refers to a promoter the nucleotide sequence of which has beenartificially altered. Nucleotide deletion(s), insertion(s) ormutation(s), alone or in combination, are examples of such artificialalterations. Artificial promoter alterations can be effected in atargeted fashion, for example by homologous recombination approaches,such as gene targeting, knockout, knock in, site-directed mutagenesis,or artificial zinc finger nuclease-mediated strategies. Alternatively,such alterations may be effected by a random or quasi-random event, suchas irradiation or non-targeted nucleotide integration and subsequentselection. Promoter modifications, in general, are fashioned in order tomodulate the transcriptional activation properties of the respectivepromoter. For example, the disruption or deletion of a regulatoryelement mediating the repression of a XYL1, XYL2, XYL3, XYLA, DGA1,ACC1, or SCD promoter in response to elevated intracellular fatty acidlevels would lead to continued transcriptional activation of therespective gene even under conditions of elevated intracellular fattyacid levels. Similarly, the insertion of a constitutively activetranscriptional activator element into a conditional promoter region mayeffect overexpression of the respective gene under normally inhibitiveconditions. Methods for the targeted disruption of a native promoter,for example, a native XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, or SCDpromoter, in a microbe, for example, for targeted disruption resultingin an increased transcription rate, are well known to those of skill inthe art.

Some aspects of this invention relate to engineering of a microbe, forexample, Y. lipolytica, to exhibit a required and/or desirable phenotypefor large-scale production of a biofuel or biofuel precursor. Someaspects of this invention relate to the metabolic engineering of thelipid synthesis pathway in order to yield a microbe optimized forbiofuel production. Some aspects of this invention relate to metabolicengineering that comprises a combination of genetic modificationsmodulating the expression of genes regulating carbon flux into a lipidsynthesis pathway in order to yield a microbe optimized for biofuelproduction. In some embodiments, the combination of geneticmodifications includes a push modification and a pull modification. Insome embodiments, the push modification comprises a genetic modificationthat increases the level of metabolites, acetyl-CoA, ATP, or NADPH forlipid synthesis in a cell, for example, overexpression of an ACC1 geneproduct. In some embodiments, the pull modification is a geneticmodification that decreases the level of a product or intermediary oflipid synthesis that exhibits a feedback inhibitory function, forexample, a fatty acid. In some embodiments, the pull modificationcomprises overexpression of a DGA1 and/or an SCD gene product.

Engineered Microbes for Biofuel Production

Some aspects of this invention relate to a microbe engineered and/oroptimized for large-scale biofuel or biofuel precursor production. Insome embodiments, an engineered microbe is provided that has beenmanipulated by a method or using a nucleic acid or protein provided bysome aspects of this invention, for example, an expression construct ora combination of expression constructs as provided herein, resulting inthe overexpression of a gene product or a combination of gene productsmediating the metabolism of a 5C sugar such as xylose, such as XYL1 andXYL2, and optionally XYL3, or XYLA. In some embodiments, an engineeredmicrobe is provided that has been manipulated by a method or using anucleic acid or protein provided by some aspects of this invention, forexample, an expression construct or a combination of expressionconstructs as provided herein, resulting in the overexpression of acombination of a gene product mediating a push process of lipidsynthesis (e.g., an ACC1 product), and a gene product mediating a pullprocess of lipid synthesis (e.g., a DGA1 and/or SCD gene product). Insome embodiments, an engineered microbe is provided, that overexpressesa push-and-pull combination of gene products that, according to someaspects of this invention, confers a required and/or desirable phenotypefor biofuel or biofuel precursor production to the microbe. In someembodiments, a microbe comprising an increased XYL1, XYL2, XYL3, XYLA,DGA1, ACC1, SCD, or ACL gene product activity is provided. In someembodiments, the microbe exhibits an increased fatty acid synthesisrate, an increased TAG storage, and/or an additional required ordesirable trait.

In some embodiments, the engineered microbe is an oleaginous yeast, forexample, Y. lipolytica. In some embodiments, an engineered yeastprovided by this invention exhibits one or more highly desirable andunexpected phenotypic characteristics, for example: increased carbon tooil conversion rate or efficiency, increased lipid accumulation in alipid body.

In some embodiments, the engineered microbe, for example, the engineeredyeast, provided by aspects of this invention exhibits a carbon to oilconversion rate within the range of about 0.02 g/g (g oil, lipid, or TAGproduced/g Glucose consumed) to about 0.3 g/g. In some embodiments, theengineered microbe, for example, the engineered yeast, provided byaspects of this invention exhibits a carbon to oil conversion of about0.010 g/g (g TAG produced/g Glucose consumed), about 0.02 g/g, about0.025 g/g, about 0.03 g/g, about 0.04 g/g, about 0.05 g/g, about 0.06g/g, about 0.07 g/g, about 0.075 g/g, about 0.08 g/g, about 0.09 g/g,about 0.1 g/g, about 0.11 g/g, about 0.12 g/g, about 0.13 g/g, about0.14 g/g, about 0.15 g/g, about 0.16 g/g, about 0.17 g/g, about 0.18g/g, about 0.19 g/g, about 0.2 g/g, about 0.21 g/g, about 0.22 g/g,about 0.23 g/g, about 0.24 g/g, about 0.25 g/g, about 0.26 g/g, about0.27 g/g, about 0.28 g/g, about 0.29 g/g, about 0.3 g/g, about 0.31 g/g,about 0.32 g/g, or approaching theoretical values. In some embodiments,the engineered microbe, for example, the engineered yeast, provided byaspects of this invention exhibits a carbon to oil conversion rate of atleast about 0.010 g/g (g TAG produced/g Glucose consumed), at leastabout 0.02 g/g, at least about 0.025 g/g, at least about 0.03 g/g, atleast about 0.04 g/g, at least about 0.05 g/g, at least about 0.06 g/g,at least about 0.07 g/g, at least about 0.075 g/g, at least about 0.08g/g, at least about 0.09 g/g, at least about 0.1 g/g, at least about0.11 g/g, at least about 0.12 g/g, at least about 0.13 g/g, at leastabout 0.14 g/g, at least about 0.15 g/g, at least about 0.16 g/g, atleast about 0.17 g/g, at least about 0.18 g/g, at least about 0.19 g/g,at least about 0.2 g/g, at least about 0.21 g/g, at least about 0.22g/g, at least about 0.23 g/g, at least about 0.24 g/g, at least about0.25 g/g, at least about 0.26 g/g, at least about 0.27 g/g, at leastabout 0.28 g/g, at least about 0.29 g/g, at least about 0.3 g/g, atleast about 0.31 g/g, at least about 0.32 g/g, or approachingtheoretical values.

Some aspects of this invention provide engineered microbes for oilproduction that can use a variety of carbon sources, including, but notlimited to fermentable sugars, for example, C5 sugars, such as xylose;C6 sugars, such as glucose; organic acids, e.g., acetic acid, and/ortheir salts, e.g., acetate; polyol compounds, such as glycerol; andsugar alcohols, such as arabitol.

Microbial Cultures for Biofuel Production

Some aspects of this invention relate to cultures of geneticallymodified microbes provided herein. In some embodiments, the culturecomprises a genetically modified microbe provided herein and a medium,for example, a liquid medium. In some embodiments, the culture comprisesa genetically modified microbe provided herein and a carbon source, forexample, a fermentable carbohydrate source, or an organic acid or saltthereof. In some embodiments, the culture comprises a geneticallymodified microbe provided herein and a salt and/or buffer establishingconditions of salinity, osmolarity, and pH, that are amenable tosurvival, growth, and/or carbohydrate to biofuel or biofuel precursorconversion by the microbe. In some embodiments, the culture comprises anadditional component, for example, an additive. Non-limiting examples ofadditives are nutrients, enzymes, amino acids, albumin, growth factors,enzyme inhibitors (for example protease inhibitors), fatty acids,lipids, hormones (e.g., dexamethasone and gibberellic acid), traceelements, inorganic compounds (e.g., reducing agents, such asmanganese), redox-regulators (e.g., antioxidants), stabilizing agents(e.g., dimethylsulfoxide), polyethylene glycol, polyvinylpyrrolidone(PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g., NaCl),chelating agents (e.g., EDTA, EGTA), and enzymes (e.g., cellulase,dispase, hyaluronidase, or DNase). In some embodiments, the culture maycomprise a drug inducing or inhibiting transcription from a conditionalor inducible promoter, for example doxicycline, tetracycline, tamoxifen,IPTG, hormones, or metal ions.

While the specific culture conditions, for example, the concentration ofthe carbon source, will depend upon the respective engineeredmicroorganism to be cultured, general methods and culture conditions forthe generation of microbial cultures are well known to those of skill inthe art, and are described, for example, in J. Sambrook and D. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd edition (Jan. 15, 2001); David C. Amberg, Daniel J. Burke;and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold SpringHarbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press(April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, andGerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A,Volume 194 (Methods in Enzymology Series, 194), Academic Press (Mar. 11,2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics andMolecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology,Vol 350), Academic Press; 1st edition (Jul. 2, 2002); and ChristineGuthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular andCell Biology, Part C, Volume 351, Academic Press; 1st edition (Jul. 9,2002), all of which are incorporated by reference herein. For oilproduction, the cultures of engineered microbes described herein arecultured under conditions suitable for oil accumulation, as known in theart.

In some embodiments, the genetically modified microbe exhibits a growthadvantage over wild type microbes of the same kind and/or over othermicrobes, for example, microbes commonly found to contaminate microbialcultures for carbon source to biofuel or biofuel precursor conversion.In some embodiments, the growth and/or proliferation advantage of anengineered microbe provided by aspects of this invention translates intothe possibility of using non-sterile culturing and fermentationconditions for biofuel or biofuel precursor production, because theproblem of culture overgrowth by contaminating microbes is mitigated orcompletely abolished. In some embodiments, an engineered microbeprovided by aspects of this invention is cultured under non-sterileconditions for biofuel or biofuel precursor production. For example, insome embodiments, non-sterilized feedstock, non-sterilized culturemedia, non-sterilized supplements, or a non-sterilized bioreactor (e.g.an open reactor under non-sterile conditions) is used for biofuel orbiofuel precursor production.

A variety of different microbes can be genetically modified according tosome aspects of this invention and used for industrial-scale biofuel orbiofuel precursor production, for example, microbes from various sourcesof yeast, such as oleaginous yeast, bacteria, algae and fungi.Non-limiting examples of suitable yeast cells are cells from Yarrowialipolytica, Hansenula polymorpha, Pichia pastoris, Saccharomycescerevisiae, S. bayanus, S. K. lactis, Waltomyces lipofer. Mortierellaalpine, Mortierella isabellina, Hansenula polymorpha., Mucor rouxii,Trichosporon cutaneu, Rhodotorula glutinis Saccharomyces diastasicus,Schwanniomyces occidentalis, S. cerevisiae, Pichia stipitis, andSchizosaccharomyces pombe. Non-limiting examples of suitable bacteriaare Bacillus subtilis, Salmonella, Escherichia coli, Vibrio cholerae,Streptomyces, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonassp, Rhodococcus sp, Streptomyces sp, and Alcaligenes sp. Non-limitingexamples of suitable fungal cells can, for example, be cultured fromspecies such as Aspergillus shirousamii, Aspergillus niger andTrichoderma reesei. Non-limiting examples of suitable algal cells arecells from Neochloris oleoabundans, Scenedesmus obliquus,Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris,Chlorella emersonii, and Spirulina maxima.

Methods for Biofuel Production/Feedstock/Bioreactors

Some aspects of this invention provide methods for the production ofbiofuel or biofuel precursors using genetically modified microbesprovided herein. In some embodiments, methods for biofuel or biofuelprecursor production on an industrial scale are provided.

A variety of carbon sources can be converted into a biofuel or biofuelprecursor using a method and/or a genetically modified microbe providedherein. In some embodiments, the carbon source comprises a carbohydrate.Sugars, starches, and fibers are non-limiting examples of carbohydratesources suitable for conversion methods provided herein. According tosome aspects of this invention, a carbohydrate source may comprise arefined and/or unrefined sugar, starch, and/or fiber, or a combinationof any of these. Non-limiting examples of sugars are fermentable sugars,such as, xylose, glucose, fructose, sucrose and lactose. Non-limitingexamples of starches are amylase and amylopectin. Non-limiting examplesof fibers are plant fibers, such as cellulose, hemicellulose and woodfibers. Some aspects of this invention relate to the use of industrialbyproducts, intermediates, or waste products, for example raw plantextracts, molasses, stover, or sewage as a carbon source. In someembodiments, the carbon source is derived from algae. In someembodiments, algal biomass is produced specifically for use as a carbonsource in microbe-mediated biofuel or biofuel precursor production.

In some embodiments, methods for the production of biofuel or biofuelprecursor are provided that include the use of a cheap, abundant, andreadily available carbon source feedstock as the carbon source. In someembodiments, cellulose or hemicellulose is used as the carbon source. Insome embodiments, the cellulose or hemicellulose is derived fromindustrial by- or waste products. In some embodiments, the cellulose orhemicellulose is derived directly from plant or algal biomass. Plant oralgal biomass is one of the most abundant feedstocks and comprises asignificant amount of non-fermentable sugars and fibers, for example,cellulose and hemi-cellulose. In some embodiments, biomass feedstock ispretreated to convert a non-fermentable sugar or fiber into afermentable sugar, thus making them available for microbe growth andmicrobe-mediated biofuel or biofuel precursor production. In someembodiments, the pretreatment of biomass feedstock includesdepolymerizing cellulose and/or hemicellulose components to monomericsugars using a pretreatment method known to those of skill in the art,for example, a dilute acid or ammonia fiber expansion (AFEX) method(see, e.g., Yang B, Wyman C E. Dilute acid and autohydrolysispretreatment. Methods Mol Biol. 2009; 581:103-14; Balan V, Bals B,Chundawat S P, Marshall D, Dale B E, Lignocellulosic biomasspretreatment using AFEX Methods Mol Biol. 2009; 581:61-77). Othermethods for depolymerization of biomass polymers to monomeric sugars arewell known to those of skill in the art and are contemplated to be usedin some embodiments of this invention.

In some embodiments, a biomass feedstock containing non-fermentablesugars is pretreated using a dilute acid method to depolymerize anon-fermentable sugar to a monomeric, fermentable sugar. In someembodiments, biomass is treated with dilute sulphuric acid at moderatelymild temperatures for a defined period of time. For example, in someembodiments, the biomass is treated with about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, or about 6% sulphuric acid. In someembodiments, the biomass is treated at about 30° C., at about 37° C., atabout 40° C., at about 50° C., at about 60° C., at about 70° C., atabout 80° C., at about 90° C., at about 100° C., at about 110° C., atabout 120° C., at about 130° C., at about 140° C., at about 150° C., atabout 175° C., at about 200° C., or at above about 200° C.

In some embodiments, the resulting hydrolysate contains insoluble ligninand solubilized cellulosic and hemicellulosic polymers. The latterproducts can be further treated to generate hexose and pentose sugarssuch as glucose and xylose monomers by methods well known to those ofskill in the art, for example, by treatment with cellulase or otherhydrolyzing enzymes. In some embodiments, the pretreatment ofnon-fermentable sugars with dilute acid results in the generation ofby-products that include toxic compounds which inhibit growth, decreaseviability, and/or inhibit biofuel or biofuel precursor production ofmicrobes not engineered according to aspects of this invention. In someembodiments, the pre-treated feedstock is washed, supplemented withmedia supporting microbial growth and biofuel or biofuel precursorproduction, and/or over-limed for detoxification.

In some embodiments, a biomass feedstock containing non-fermentablesugars is pretreated using an AFEX method to depolymerize anon-fermentable sugar to a monomeric, fermentable sugar. In someembodiments, biomass is treated with liquid ammonia at high temperatureand pressure for a defined period of time. In some embodiments, biomassis treated for about 10 minutes, about 20 minutes, about 30 minutes,about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes,about 80 minutes, about 90 minutes, or longer. In some embodiments,biomass is treated at about 30° C., at about 37° C., at about 40° C., atabout 50° C., at about 60° C., at about 70° C., at about 80° C., atabout 90° C., at about 100° C., at about 110° C., at about 120° C., atabout 130° C., at about 140° C., at about 150° C., at about 175° C., atabout 200° C., or at above about 200° C. In some embodiments, the AFEXpretreatment results in the conversion of crystalline cellulosecontained in the feedstock into an amorphous, fermentable form. In someembodiments, the AFEX pre-treated biomass feedstock does not containsignificant amounts of toxic byproducts that inhibit microbial growthand/or biofuel or biofuel precursor production, and is used withoutprior detoxification for microbial biofuel or biofuel precursorproduction.

In some embodiments, biomass feedstock, with or without pre-treatment,is treated with an enzyme that hydrolyzes or depolymerizes sugarpolymers, for example, with a cellulase or hemicellulase enzyme. In someembodiments, the feedstock is contacted with the enzyme in a liquidphase and incubated at a temperature allowing for the enzyme to catalyzea depolymerization or hydrolyzation reaction for a time sufficient tohydrolyze or depolymerize a significant amount of the non-fermentablesugar or fiber in the biomass feedstock. In some embodiments, the liquidphase of the feedstock contacted with the enzyme, which contains thesoluble, fermentable sugar fraction, is separated from the solid phase,including non-fermentable sugars and fibers, after incubation forhydrolyzation and depolymerization, for example, by centrifugation. Insome embodiments, the liquid fraction of the feedstock is subsequentlycontacted with a microbe, for example, a microbe provided by aspects ofthis invention, for conversion to biofuel or biofuel precursor. In someembodiments, enzymatic conversion of non-fermentable sugars or fiberoccurs in a consolidated bioprocess, for example, at the same timeand/or in the same reactor as microbial conversion of the producedfermentable sugars to biofuel or biofuel precursor. In some embodiments,the enzymatic conversion is performed first, and the feedstock contactedwith enzyme is subsequently contacted with the microbe for biofuel orbiofuel precursor production. In some embodiments, enzymatic andmicrobial conversion are performed at the same time and in the samereactor.

In some embodiments, an engineered microbe as provided herein, forexample, a Yarrowia lipolytica overexpressing a XYL1, XYL2, XYL3, XYLA,DGA1, ACC1, SCD, or ACL gene product, is grown on glycerol. In someembodiments, the genetically modified microbes are intermittentlycontacted with glycerol. In some embodiments, the microbes arecontinuously or semi-continuously contacted with glycerol. In someembodiments, the microbes are contacted with glycerol at a concentrationof about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%vol/vol. Contacting the engineered microbes provided herein withglycerol provides metabolites for the production of TAGs, as well asreducing moieties for the production of fatty acids from carbohydrates.In some embodiments, glycerol spiking or use is performed in biofuel orbiofuel precursor production methods in combination with any othercarbon source described herein.

In some embodiments, fermentation processes for large-scalemicrobe-mediated carbohydrate to lipid conversion may be carried out inbioreactors. As used herein, the terms “bioreactor” and “fermentor,”which are interchangeably used, refer to an enclosure, or partialenclosure, in which a biological and/or chemical reaction takes place,at least part of which involves a living organism or part of a livingorganism. A “large-scale bioreactor” or “industrial-scale bioreactor” isa bioreactor that is used to generate a product, for example a biofuelor biofuel precursor, for example a fatty acid and/or TAG, on acommercial or quasi-commercial scale. Large scale bioreactors typicallyhave volumes in the range of liters, hundreds of liters, thousands ofliters, or more.

A bioreactor in accordance with aspects of this invention may comprise amicrobe or a microbe culture. In some embodiments, a bioreactor maycomprise a spore and/or any kind of dormant cell type of any isolatedmicrobe provided by aspects of this invention, for example, in a drystate. In some embodiments, addition of a suitable carbohydrate sourceto such bioreactors may lead to activation of the dormant cell, forexample to germination of a yeast spore, and subsequent conversion ofthe carbohydrate source, at least in part, to a biofuel or biofuelprecursor.

Some bioreactors according to aspects of this invention may include cellculture systems where microbes are in contact with moving liquids and/orgas bubbles. Microbes or microbe cultures in accordance with aspects ofthis invention may be grown in suspension or attached to solid phasecarriers. Non-limiting examples of carrier systems include microcarriers(e.g., polymer spheres, microbeads, and microdisks that can be porous ornonporous), cross-linked beads (e.g., dextran) charged with specificchemical groups (e.g., tertiary amine groups), 2D microcarriersincluding cells trapped in nonporous polymer fibers, 3D carriers (e.g.,carrier fibers, hollow fibers, multicartridge reactors, andsemi-permeable membranes that can comprising porous fibers),microcarriers having reduced ion exchange capacity, encapsulation cells,capillaries, and aggregates. Carriers can be fabricated from materialssuch as dextran, gelatin, glass, and cellulose.

Industrial-scale carbohydrate to lipid conversion processes inaccordance with aspects of this invention may be operated in continuous,semi-continuous or non-continuous modes. Non-limiting examples ofoperation modes in accordance with this invention are batch, fed batch,extended batch, repetitive batch, draw/fill, rotating-wall, spinningflask, and/or perfusion mode of operation.

In some embodiments, bioreactors may be used that allow continuous orsemi-continuous replenishment of the substrate stock, for example acarbohydrate source and/or continuous or semi-continuous separation ofthe product, for example a secreted lipid, an organic phase comprising alipid, and/or cells exhibiting a desired lipid content, from thereactor.

Non-limiting examples of bioreactors in accordance with this inventionare: stirred tank fermentors, bioreactors agitated by rotating mixingdevices, chemostats, bioreactors agitated by shaking devices, airliftfermentors, packed-bed reactors, fixed-bed reactors, fluidized bedbioreactors, bioreactors employing wave induced agitation, centrifugalbioreactors, roller bottles, and hollow fiber bioreactors, rollerapparatuses (for example benchtop, cart-mounted, and/or automatedvarieties), vertically-stacked plates, spinner flasks, stirring orrocking flasks, shaken multiwell plates, MD bottles, T-flasks, Rouxbottles, multiple-surface tissue culture propagators, modifiedfermentors, and coated beads (e.g., beads coated with serum proteins,nitrocellulose, or carboxymethyl cellulose to prevent cell attachment).

Bioreactors and fermentors according to aspects of this invention may,optionally, comprise a sensor and/or a control system to measure and/oradjust reaction parameters. Non-limiting examples of reaction parametersare: biological parameters, for example growth rate, cell size, cellnumber, cell density, cell type, or cell state, chemical parameters, forexample pH, redox-potential, concentration of reaction substrate and/orproduct, concentration of dissolved gases, such as oxygen concentrationand CO2 concentration, nutrient concentrations, metaboliteconcentrations, glucose concentration, glutamine concentration, pyruvateconcentration, apatite concentration, concentration of an oligopeptide,concentration of an amino acid, concentration of a vitamin,concentration of a hormone, concentration of an additive, serumconcentration, ionic strength, concentration of an ion, relativehumidity, molarity, osmolarity, concentration of other chemicals, forexample buffering agents, adjuvants, or reaction by-products,physical/mechanical parameters, for example density, conductivity,degree of agitation, pressure, and flow rate, shear stress, shear rate,viscosity, color, turbidity, light absorption, mixing rate, conversionrate, as well as thermodynamic parameters, such as temperature, lightintensity/quality etc.

Sensors able to measure parameters as described herein are well known tothose of skill in the relevant mechanical and electronic arts. Controlsystems able to adjust the parameters in a bioreactor based on theinputs from a sensor as described herein are well known to those ofskill in the art of bioreactor engineering.

The type of carbon source to be employed for conversion to a biofuel orbiofuel precursor according to aspects of this invention depends on thespecific microbe employed. Some microbes provided by aspects of thisinvention may be able to efficiently convert a specific carbohydratesource, while a different carbohydrate source may not be processed bythe same microbe at high efficiency or at all. According to aspects ofthis invention, the modified oleaginous yeast Y. lipolytica, forexample, can efficiently convert sugars, such as xylose, glucose,fructose, sucrose, and/or lactose, and carbohydrate sources high insugars, for example molasses, other carbon sources such as glycerol andarabitol, and plant fibers into fatty acids and their derivatives.

In some embodiments, a biofuel or biofuel precursor, for example, afatty acid or a triacylglycerol, generated from a carbon sourcefeedstock is secreted, at least partially, by a microbe provided byaspects of this invention, for example, an oleaginous yeast, such as aY. lipolytica cell. In some embodiments, a microbe provided by aspectsof this invention is contacted with a carbohydrate source in an aqueoussolution in a bioreactor, and secreted biofuel or biofuel precursorforms an organic phase that can be separated from the aqueous phase. Theterm organic phase, as used herein, refers to a liquid phase comprisinga non-polar, organic compound, for example a fatty acid, TAG, and/orother non-polar lipid. And organic phase in accordance to this inventionmight further contain a microbe, a carbohydrate, or other compound foundin other phases found in a respective bioreactor. Methods useful forindustrial scale phase separation are well known to those of ordinaryskill in the art. In some embodiments, the organic phase is continuouslyor semi-continuously siphoned off. In some embodiments, a bioreactor isemployed, comprising a separator, which continuously orsemi-continuously extracts the organic phase.

In some embodiments, a biofuel or biofuel precursor is accumulated incells according to aspects of this invention. In some embodiments, cellsthat have accumulated a desirable amount of biofuel or biofuelprecursor, are separated continuously or semi-continuously from abioreactor, for example, by centrifugation, sedimentation, orfiltration. Cell separation can further be effected, for example, basedon a change in physical cell characteristics, such as cell size ordensity, by methods well known to those skilled in the art. Theaccumulated biofuel or biofuel precursor can subsequently be extractedfrom the respective cells using standard methods of extraction wellknown to those skilled in the art, for example, solvent hexaneextraction. In some embodiments, microbial cells are collected andextracted with 3 times the collected cell volume of hexane. In someembodiments, the extracted biofuel or biofuel precursor are furtherrefined. In some embodiments, a biofuel precursor, for example atriacylglycerol is converted to a biofuel, for example, biodiesel, usinga method well known to those of skill in the art, for example, atransesterification procedure.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples below. Thefollowing examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.Accordingly, it will be understood that the example section is not meantto limit the scope of the invention.

EXAMPLES Example 1 Engineering Xylose Utilization in the OleaginousYeast Yarrowia lipolytica for Biofuel Production

Introduction

In the search for improved feedstocks, the push towards cellulosicbiofuels is a clear choice. Cellulosic biomass mitigates the need tocompete with food crop production; an estimated 1.3+ billion dry tonsper year of biomass is potentially available in the US alone (Perlack2005). Additionally, cellulosic materials can be more efficiently grownand more stably produced compared to sugar crops. However cellulosicmaterials are not naturally consumable by most biofuel-producingorganisms, and thus cellulose requires pretreatment and hydrolysis tobreak the material down into monomeric sugar. The resulting hydrolysatecan then be used as a sugar rich feedstock.

Since hydrolysis of lignocellulosic biomass results in 20-30%carbohydrates in the form of xylose, utilization of pentose sugars isone of the first steps toward efficiently using cellulosic materials.Saccharomyces cerevisiae, the most productive of ethanologenicorganisms, cannot ferment xylose; it lacks the ability to convert xyloseinto xylulose, which can then enter the pentose phosphate pathway (PPP).Transferring the xylose reductase (XR or XYL1) and xylitol dehydrogenase(XDH or XYL2) enzymes from Scheffersomyces stipitis (formerly Pichiastipitis) has been shown to enable growth of the yeast on xylose forproduction of ethanol (Jeffries 2006). The addition of xylulokinase (XKor XYL3) can also be used to further improve utilization, although S.cerevisiae already carries an endogenous version of this gene. Asecondary pathway, using xylose isomerase (XYLA), can be used to convertxylose into xylulose. Compared to the XR/XDH redox pathway, which usesNADPH and NAD+ cofactors for shuttling of reducing equivalents, theisomerase pathway requires no cofactors. Nonetheless the redox pathwayis much more prevalent in nature, and likewise in literature (Jeffries2006; Matsushika et al. 2009).

Instead of ethanol production, it may also be advantageous to produceyeast oil for biodiesel from cellulosic feedstocks. As a robust lipidproducing organism, Yarrowia lipolytica appears to be an attractiveplatform for the production of cellulosic biodiesel. By leveraging theknowledge and resources developed for xylose metabolic engineering in S.cerevisiae, xylose utilization in Y. lipolytica enables robustproduction of yeast oils from cellulosic materials. Because theoreticalyields of lipid production from xylose are very similar to that ofglucose (0.34 g/g compared to 0.32 g/g), the consumption of xyloserepresents an attractive and worthwhile opportunity in a developingcellulosic biodiesel microbial bioprocess (Ratledge 1988). Furthermore,Y. lipolytica has a very high relative PPP flux (Blank et al. 2005), aphenotype advantageous for growth on xylose since all flux must passthrough the PPP. Upregulation of the PPP pathway is a commonlyengineered aspect in xylose utilizing S. cerevisiae strains (Walfridssonet al. 1995).

For the metabolic conversion of xylose to lipids, xylose enters the celland can be catabolized either through the redox (XR/XDH) pathway or theisomerase (XYLA) pathway, producing xylulose. It can then enter centralmetabolism through the non-oxidative pathway of the PPP where itultimately produces glyceraldehyde-3-phosphate (G3P) andfructose-6-phosphate (F6P). These two products can then enter the restof central metabolism, going through glycolysis to enter the TCA cycle.Production of lipids occurs normally through the transport ofmitochondrial citrate into the cytosol, where it is cleaved by ATPcitrate lyase into oxaloacetate and cytosolic acetyl-coA. The acetyl-coAcan then enter the fatty acid synthesis pathway through the enzymaticactivity of acetyl-coA carboxylase. Acyl-coA generated from the fattyacid synthase complex are transferred to a glycerol-3-phosphate backboneand ultimately sequestered within lipid bodies as triacylglycerol (TAG).

Here we describe the analysis of Y. lipolytica for its natural xyloseutilization and the metabolic engineering of the organism enablingutilization of xylose for the production of lipids. By incorporation ofXR/XDH genes we are able to enable growth on xylose as sole carbonsource, and open up opportunities for the production of lipids fromcofermentations. Next we study the performance of our engineered strainthrough the use of cofermentations to analyze for catabolite repressionand response, and evaluate the performance of the strain in a scaled-up2-L bioreactor glycerol-xylose cofermentation with respect to lipidproduction. Finally we perform transcription analysis to observe therespiratory responses of the organism during cofermentation.

Methods

Yeast Strains, Growth, and Culture Conditions

The Y. lipolytica strains used in this study were derived from thewild-type Y. lipolytica W29 strain (ATCC20460). The auxotrophic Po1g(Leu-) used in all transformations was obtained from Yeastern BiotechCompany (Taipei, Taiwan). All strains used in this study are listed inTable 1. Constructed plasmids were linearized with SacII andchromosomally integrated into Po1g according to the one-step lithiumacetate transformation method described by Chen et al. (Chen et al.,1997). MTYL transformants were named after the numbering of theircorresponding integrated plasmids. Transformants were plated onselective media and verified by PCR of prepared genomic DNA. Verifiedtransformants were then stored as frozen glycerol stocks at −80° C. andon selective YNB plates at 4° C.

Media and growth conditions for Escherichia coli have been previouslydescribed by Sambrook et al. (Sambrook and Russell 2001), and those forY. lipolytica have been described by Barth and Gaillardin (Barth andGaillardin 1997). Rich medium (YPD) was prepared with 20 g/L Bactopeptone (Difco Laboratories, Detroit, Mich.), 10 g/L yeast extract(Difco), 20 g/L glucose (Sigma-Aldrich, St. Louis, Mo.). YNB medium wasmade with 1.7 g/L yeast nitrogen base (without amino acids) (Difco),0.69 g/L CSM-Leu (MP Biomedicals, Solon, Ohio), and 20 g/L glucose.Selective YNB plates contained 1.7 g/L yeast nitrogen base (withoutamino acids), 0.69 g/L CSM-Leu, 20 g/L glucose, and 15 g/L Bacto agar(Difco).

Shake flask experiments were carried out using the following medium: 1.7g/L yeast nitrogen base (without amino acids), 1.5 g/L yeast extract,and 50 g/L glucose. From frozen stocks, precultures were inoculated intoYNB medium (5 mL in Falcon tube, 200 rpm, 28° C., 24 hr). Overnightcultures grown in YPD were centrifuged, washed, and reinoculated into 50mL of media in 250 mL Erlenmeyer shake flask (200 rpm, 28° C.). OD,biomass and sugar content were taken periodically and analyzed.

For adaptation of strains on xylose, verified transformants wereinoculated into shake flasks containing minimal media and 20 g/L xylose.The cultures were incubated at 30° C. for at least 10 days, waiting forgrowth to occur, before reinoculation into fresh media. This process wasrepeated until the final OD of the culture reached at least 20,indicating adaptation to xylose. The culture was then stored as frozenstock in 15% glycerol at −80° C. for subsequent use.

Bioreactor scale fermentation was carried out in a 2-liter baffledstirred-tank bioreactor. The medium used contained 1.7 g/L yeastnitrogen base (without amino acids and ammonium sulfate), 2 g/L ammoniumsulfate, 1 g/L yeast extract, and 90 g/L glucose. From a selectiveplate, an initial preculture was inoculated into YPD medium (40 mL in250 mL Erlenmeyer flask, 200 rpm, 28° C., 24 hr). Exponentially growingcells from the overnight preculture were transferred into the bioreactorto an optical density (A600) of 0.1 in the 2-L reactor (2.5 vvmaeration, pH 6.8, 28° C., 250 rpm agitation). Time point samples werestored at −20° C. for subsequent lipid analysis. Sugar organic acidcontent was determined by HPLC. Biomass was determined by determinedgravimetrically from samples washed and dried at 60° C. for two nights.Lipid content was analyzed by direct transesterification.

Plasmid Construction

Standard molecular genetic techniques were used throughout this study(Sambrook and Russell 2001). Restriction enzymes and PhusionHigh-Fidelity DNA polymerase used in cloning were obtained from NewEngland Biolabs (Ipswich, Mass.). Genomic DNA from yeast transformantswas prepared using Yeastar Genomic DNA kit (Zymo Research, Irvine,Calif.). All constructed plasmids were verified by sequencing. PCRproducts and DNA fragments were purified with PCR Purification Kit orQIAEX II kit (Qiagen, Valencia, Calif.). Plasmids used are described inTable 1. Primers used are described in Table 2.

Plasmid pMT041 was constructed by amplifying the xylose reductase gene(XYL1; Accession Number: XM_001385144) from S. stipitis genomic DNA(ATCC 58376) using the primers MT243 and MT244 and inserting it betweenthe Pm1I and BamHI sites of pINA1269. Plasmid pMT044 was constructed byamplifying the xylitol dehydrogenase gene (XYL2; Accession Number:XM_001386945) using the primers MT233 and MT234 and inserting it betweenthe Pm1I and BamHI sites of pINA1269. XYL1 and XYL2 are both genesoriginally from the xylose utilizing yeast, S. stipitis.

Plasmid pMT059 was constructed by amplifying the XYL1 gene from pMT041using the primers MT281 and MT282. The amplicon was then inserted intothe TEFin expression plasmid, pMT015 between the sites SnaBI and KpnI.

For the expression of multiple genes on a single plasmid, thepromoter-gene-terminator cassette can be amplified from a parent vectorusing primers MT220 and MT265. The cassette can then be inserted intothe receiving vector between the restriction sites NruI and AseI,resulting in a tandem gene construct. The AseI restriction site wasselected to facilitate selection, as it resides within the ampicillinresistance marker of the plasmid. Because NruI is a blunt endrestriction site, insertion of the amplicon does not increase the totalnumber of NruI sites that helps facilitate progressive insertions.Plasmid pMT081 was constructed by amplifying the XYL2 cassette frompMT044 and inserting it into the plasmid pMT059, containing XYL1.Plasmid pMT085 was constructed by amplifying the DGA cassette frompMT053 and inserting it into the plasmid pMT081, which contains XYL12.

RNA Isolation and Transcript Quantification

Shake flask cultures grown for 42 hrs were collected and centrifuged for5 min at 10,000 g. Each pellet was resuspended in 1.0 ml of Trizolreagent (Invitrogen) and 100 μL of acid-washed glass beads were added(Sigma-Aldrich). Tubes were vortexed for 15 min at 4° C. for cell lysisto occur. The tubes were then centrifuged for 10 min at 12,000 g at 4°C. and the supernatant was collected in a fresh 2-mL tube. 200 μLchloroform was then added and tubes were shaken by hand for 10 seconds.The tubes were again centrifuged for 10 min at 12,000 g at 4° C. 400 μLof the upper aqueous phase was transferred to a new tube, and an equalvolume of phenol-chloroform-isoamyl alcohol (pH 4.7) (Ambion, Austin,Tex.) was added. Tubes were again shaken by hand for 10 seconds andcentrifuged for 10 min at 12,000 g at 4° C. 250 μL of the upper phasewas transferred to a new tube with an equal volume of cold ethanol and1/10th volume sodium acetate (pH 5.2). Tubes were chilled at −20° C. forthirty minutes to promote precipitation. Tubes were then centrifuged for5 min at 12,000 g, washed twice with 70% ethanol, dried in a 60° C. ovenand finally resuspended in RNAse free water. RNA quantity was analyzedusing a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, Del.) and samples were stored in −80° C. freezer. qRT-PCRanalyses were carried out using iScript One-step RT-PCR Kit with SYBRGreen (Bio-Rad, Hercules, Calif.) using the Bio-Rad iCycler iQ Real-TimePCR Detection System. Fluorescence results were analyzed using Real-timePCR Miner and relative quantification and statistical analysis wasdetermined with REST 2009 (Qiagen) using actin as the reference gene andMTYL038 as the reference strain (Zhao and Fernald 2005). Samples wereanalyzed in quadruplicate.

TABLE 1 Strains and plasmids used in this study Strains (host strain)Genotype or plasmid Source E. coli DH5α fhuA2 Δ(argF-lacZ)U169 phoAglnV44 Φ80 Δ(lacZ)M15 gyrA96 Invitrogen recA1 relA1 endA1 thi-1 hsdR17pINA1269 JMP62-LEU Yeastern pMT015 pINA1269 php4d::TEFin This ExamplepMT041 hp4d-XYL1 This Example pMT044 hp4d-XYL2 This Example pMT053YTEFin-DGA1 This Example pMT059 TEFin-XYL1 This Example pMT081TEFin-XYL1 + hp4d-XYL2 This Example pMT085 TEFin-XYL1 + hp4d-XYL2 +TEFin-DGA This Example Y. lipolytica Po1g MATa, leu2-270,ura3-302::URA3, xpr2-332, axp-2 Yeastern MTYL038 MATa, leu2-270,ura3-302::URA3, xpr2-332, axp-2 TEF-LacZ- This Example LEU2 MTYL053MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 TEFin-DGA1- This ExampleLEU2 MTYL081 MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 TEFin-LacZ-This Example LEU2 MTYL085 MATa, leu2-270, ura3-302::URA3, xpr2-332,axp-2 hp4d-ACC1- This Example LEU2

TABLE 2 Primers used in this study. SEQ Descrip- ID Primer tion NOSequence PCR MT233 XYL2 22 AATGACTGCTAACCCTTCCTTGGTGT MT234 XYL2 23CTGGTCTAGGT

TTACTCAGGGCCGT CAATGAGAC MT243 XYL1 24 AATGCCTTCTATTAAGTTGAACTCTGGTTACMT244 XYL1 25 CTAGGTCTTACT

TTAGACGAAGATA GGAATCTTGTCCCA MT281 XYL1 26TAACCGCAGCATCATCACCATCACCACCCTT CTATTAAGTTGAACTCTGGTTACGAC MT282 XYL1 27CTTACA

TTAGACGAAGATAGGAATC TTGTCCCAG RT-PCR MTR001 Actin 28 TCCAGGCCGTCCTCTCCCMTR002 Actin 29 GGCCAGCCATATCGAGTCGCA MTR017 ylXYL1 30AAGGAGTGGGCTGGATGGA MTR018 ylXYL1 31 GGTCTCTCGGGTAGGGATCTTG MTR019ylXYL2 32 ATGGAGGAATCGGCGACTT MTR020 ylXYL2 33 ACCACCTCTCCGGCACTTTMTR031 DGA 34 AACGGAGGAGTGGTCAAGCGA MTR032 DGA 35 TTATGGGGAAGTAGCGGCCAAMTR051 psXYL2 36 CTCCAAGTTGGGTTCCGTTGC MTR052 psXYL2 37GCGACAGCAGCAGCCAAAAGA MTR053 psXYL1 38 AGGCTATCGCTGCTAAGCACGG MTR054psXYL1 39 TTTGGAATGATGGCAATGCCTC MTR055 ylXYL3 40 CAGCTCAAGGGCATCATTCTGGMTR056 ylXYL3 41 TGCGGCAAGTCGTCCTCAAA MTR060 IDH1 42CTTCGAACCGCCTACCTGGCTA MTR061 IDH1 43 TGGGCTGGAACATGGTTCGA MTR064 ACO144 CACCGCTTTCGCCATTGCT MTR065 ACO1 45 GGGCTCCTTGAGCTTGAACTCC MTR066 PDB146 CTGTGGTGTCGTCAACGACTCC MTR067 PDB1 47 GCTCAATGGCGTAAGGAGTGG MTR072ICL 48 TACTCTCCCGAGGACATTGCC MTR073 ICL 49 CAGCTTGAAGAGCTTGTCAGCCRelevant restriction sites are in bold.

Direct Transesterification

For routine lipid quantification to determine relative lipidaccumulation, a method for direct transesterification of cell biomasswas used, adapted from the two-step base-then-acid-catalyzed directtransesterification method developed by Griffiths et al. (Griffiths etal. 2010). A normalized quantity of cell culture was centrifuged and themedia supernatant was removed. Samples were then stored in −20° C.freezer or directly transesterified. The cell was then resuspended withthe addition of 100 μL of hexane containing 10 mg/mL methyl tridecanoateinternal standard. 500 μL 0.5 N sodium methoxide, prepared by theaddition of sodium hydroxide to methanol, was then added to the sample.The sample was then vortexed for 1 hour at room temperature. Next 40 μLof sulfuric acid was carefully added to the sample, followed by theaddition of 500 μL of neat hexane. The sample was again vortexed at roomtemperature for another 30 minutes. 300 μL of the upper hexane layer wasthen transferred into a glass vial and run using the GC-FID, understandard operating conditions. Total lipid content was calculated as thesum of total fatty acid content for the five primary FAMEs identified.

Results & Discussion

Elucidating Endogenous Functionality of the Xylose Utilization Pathwayin Y. lipolytica

Within the literature, there are conflicting reports about the abilityfor Y. lipolytica to naturally consume xylose. In most reports, growthon xylose has not been observed (Pan et al. 2009; Ruiz-Herrera andSentandreu 2002). However, there are reports of Y. lipolytica positivelygrowing on xylose: strain Po1g was found to consume xylose in a canehydrolysate fermentation (Tsigie et al. 2011), and two strains of Y.lipolytica were grown on xylose to measure xylulose-5-phosphatephosphoketolase activity (Evans and Ratledge 1984). Beyond theseincidences, there is otherwise very little reported evidence of using Y.lipolytica for growth on xylose, despite the volume of research of usingthe organism grow on other alternative and residual substrate sources(Papanikolaou et al. 2002; Papanikolaou et al. 2003; Scioli and Vollaro1997). Table 3 lists putative XR/XDH/XK genes within the genome of Y.lipolytica from a BLAST comparison to known functional pathway genes.While the amino acid identity is only 40-52%, the expect value indicatessignificant likelihood of similarity, and Y. lipolytica often managesonly 40-60% amino acid identity with orthologous genes from S.cerevisiae, due to distal phylogeny. Nonetheless, the low homology callsinto question the potential functional characteristics of these genes,which further adds to the controversy.

TABLE 3 BLAST results for endogenous xylose utilization pathway in Y.lipolytica. Amino acid identity is indicated in comparison with theparent sequence (organism indicated in parentheses). Expect value is thestatistical false-positive rate. Accession Expect Function NumberIdentity Value Xylose reductase (XR) YALI0D07634p 49% (S. stipitis)3e−80 Xylitol dehydrogenase YALI0E12463p 52% (S. stipitis) 1e−96 (XDH)Xylulokinase (XK) YALI0F10923p 40% (S. cerevisiae) 1e−96

To test the ability for Y. lipolytica to utilize its endogenous putativeXYL123 pathway in laboratory conditions, control strain MTYL038 wasgrown in minimal media on three different substrates: xylose, xylitol,arabitol. As seen in FIG. 1A, these three substrates can be used todiagnose the functionality of the three XYL123 genes. For example,growth on xylitol will demonstrate that XYL2 and XYL3 are functional,while growth on arabitol demonstrates that XYL3 is functional. FIG. 1Bdepicts the growth curves of MTYL038 on the various substrates, with ashake flask with no carbon substrate as the control. While it was foundthat the strain did not grow on xylose, it was found to grow weakly onxylitol and quite robustly on arabitol. This suggests that while XYL1,and most likely XYL2, are not naturally expressed or functional in Y.lipolytica in the presence of their respective substrates, XYL3 isexpressed and the organism can grow utilizing this pathway as itsprimary catabolic pathway.

Expression of XYL12 Enables Growth on Xylose

With the knowledge that the endogenous xylulokinase is functional in Y.lipolytica, the remaining elements of the xylose utilization pathwaywere integrated to enable growth on xylose. The XYL1 and XYL2 genes fromS. stipitis cloned into Y. lipolytica expression cassettes. XYL1 wascloned under the control of the stronger TEFin promoter, while the XYL2gene was cloned under the control of hp4d. The XYL2 expression cassettewas inserted into the XYL1 plasmid, creating plasmid pMT081, expressingboth XYL1 and XYL2. Transformation of this plasmid into backgroundstrain Po1g yielded the strain MTYL081.

Numerous experiments working with S. cerevisiae and the xyloseutilization pathway have discovered that it is often necessary toinclude periods of adaptation—where serial dilution in xylose media isperformed—for development of stable xylose utilization (Jeffries 2006;Kuyper et al. 2004; Tomás-Pejó et al. 2010). This was similarly found tobe the case in Y. lipolytica—the verified transformant MTYL081 initiallydid not grow on xylose. It was grown in minimal xylose media in a shakeflask for 10 days before reinoculating in fresh media. This serialdilution was repeated until there was an observed increase in maximum ODto above 15. FIG. 2A shows the growth curve on the third serial dilutioncompared to the original unadapted strain and a control strain thatunderwent serial dilution in xylose media. Lack of growth from thelatter two strains shows that adaptation is necessary for xyloseutilization and adaptation does not occur in strains lacking theheterologous XYL12 genes. Adapted growth was found to be steady androughly exponential, with the maximum OD of 38 being reached after 130hours. The doubling time is roughly 25 hrs, which is significantly lowerthan rates typically observed on glucose but comparable to that onarabitol (see FIG. 1B).

To explore the underlying adaptations that improved the xylose-utilizingphenotype, RT-PCR was performed comparing the expression ofheterologously expressed XYL12 and endogenous XYL123 genes in theadapted and unadapted strains. FIG. 2B shows the relative change intranscription level of the genes after adaptation. The heterologouslyexpressed XYL1 was overexpressed 300-fold compared to the unadaptedstrain, while XYL2 was upregulated 17-fold. Within the adapted strain,XYL1 was expressed 6-fold greater than XYL2, which is in agreement withthe expression expected from the promoters used. Endogenous XYL123 wasnot significantly upregulated both in adapted MTYL081 and the controlstrain that underwent serial dilution, indicating that the observedadaptation to xylose was not an activation of the putative native xylosepathway. The strong upregulation of XYL1 and XYL2 has been similarlyobserved in metabolic engineering of S. cerevisiae, as the utilizationpathway, being both heterologously expressed and potentially therate-limiting step, requires strong overexpression for sufficient growth(Karhumaa et al. 2005; Karhumaa et al. 2007). This seems to likewise bethe case in Y. lipolytica, as the two XYL12 steps achieve very strongoverexpression and yet still only achieve a relatively low growth rate.However, it may also be that with the adapted XYL12 expression, newrate-limiting steps appear to hinder specific growth on xylose, such asPPP activity or pentose transport (Karhumaa et al. 2005).

The normal combined activity of XYL1 and XYL2 consumes one NADPH andgenerates one NADH. Without suitable means to regenerate NADPH fromNADH, this can lead to cofactor imbalances and has been seen as asignificant challenge in metabolic engineering of S. cerevisiae(Matsushika et al. 2009). However, with a potential cofactor imbalance,one would expect early cessation of growth and large accumulation ofxylitol due to complete depletion on NADPH. In our shake flask cultureswe observed only <0.5 g/L xylitol formation after consumption of 32 g/Lof xylose, while the maximum OD was very higher compared to what istypically observed in shake flasks, suggesting that cofactor balance maynot be an issue in this situation. While this does not remove thepossibility of rate-limiting steps in the exchange of NADPH to NADH,thus slowing but not stopping growth, in the presence of oxygen,mitochondrial function actively controls and maintains the NADPH/NADHequilibrium and exchange fluxes (Singh and Mishra 1995).

Cofermentation of Two Substrates for Improved Productivity

While metabolic engineering allowed growth on xylose in Y. lipolytica,growth was dramatically slower than on glucose. Possible factorscontributing to the limited growth and productivity are the lack ofdedicated pentose transporters, low PPP flux, and inability for the cellto identify xylose as a fermentable sugar (Jeffries 2006; Jin et al.2004; Matsushika et al. 2009). To improve productivities with thelimited specific growth on xylose, experiments were performed usingtwo-substrate cofermentations. Cellulosic materials typically consist ofa blend of both hexose and pentose sugars, and rarely consist of purepentose (Lee et al. 2007). Furthermore, substrates like glycerol are abyproduct of biodiesel production, and may be recycled back into theprocess. First it was necessary to characterize and determine whichcofermentation combinations are ideal for lipid production. Xylose wascombined with a helper substrate—glucose, glycerol, or arabitol—andgrown in shake flasks to determine growth characteristics and observecatabolite repression effects in the cofermentation system. Cataboliterepression is the preferential uptake of one substrate through therepression of the utilization pathway of secondary substrates, and canbe seen in a wide range of cofermentations in Y. lipolytica (Morgunovand Kamzolova 2011). The strain MTYL085 was used, which contains theXYL12 pathway as well as DGA overexpression. DGA overexpression iscapable of improving lipid accumulation and was found to be a strongcontributor to engineered lipid overproduction (Kamisaka et al. 2007).By combining both the xylose utilization pathway and elements for lipidoverproduction, we may be able to direct flux from xylose towards lipidsfor a cellulosic biodiesel platform.

FIGS. 3A-3C depict the growth characteristics and depletion of bothsubstrates for the three cofermentation combinations. For glycerol (FIG.3B), diauxic shift is clearly observed, with glycerol being consumedrapidly before any xylose is depleted. For glucose (FIG. 3A), diauxicshift was less observable, as it is possible that at very lowconcentrations of glucose, catabolite repression is weak (Morgunov andKamzolova 2011). At higher glucose concentrations, diauxic shift wasclearly observable (data not shown). While all three cultures began with4 g/L of the helper substrate, glycerol was converted into the mostbiomass after it was completely depleted, achieving an OD of 8 within 24hrs. Glycerol has been known to be a highly preferred substrate for Y.lipolytica, and unlike S. cerevisiae, there is no loss in specificgrowth rate when growing on glycerol compared to glucose (Taccari et al.2012). It is also Crabtree-negative, an effect that eschews therespiration-dependent nature of glycerol metabolism found in S.cerevisiae (De Deken 1966). As a result, MTYL085 is able to consumeslightly more xylose by the end of the culture. The evidence of diauxicshift also indicates that while the xylose uptake rate may be constantwhen grown solely on xylose, other factors must be at play in repressingthe utilization, most conspicuously pentose transport. There is agrowing body of evidence that pentose transport is a key rate-limitingstep in xylose utilization and may also be a strong contributing factortowards diauxic shift (Young et al. 2012).

The cofermentation of xylose and arabitol exhibits a much differentresponse (FIG. 3C). Since arabitol shares the same catabolic route forall but the initial pathway, it is likely the arabitol response will bemost similar to the xylose growth phenotype. Furthermore, xylosedepletion begins well before arabitol is consumed, exhibitingsimultaneous utilization of both substrates. The smooth growth profilein this case is in contrast to the two-phase growth seen in glucose orglycerol—a product of diauxic growth. Nonetheless the overall growthrate and productivity is significantly lower than glucose or glycerol.Additionally, arabitol is not a common substrate in cellulosic materialand would thus be a prohibitive cost to supplement as a feedstock.

Lipid Production in Xylose and Glycerol Cofermentation

Because glycerol showed the greatest promise for increased productivity,a scale-up cofermentation was performed using glycerol and xylose assubstrates. A 2-L bioreactor was initially charged with 20 g/L glyceroland 80 g/L xylose. The C/N ratio of the reactor was adjusted to be 100,which results nitrogen-limited conditions favorable for lipidaccumulation. The results of the fermentation are found in FIG. 4. Overthe course of 230 hrs, all the carbon substrate was consumed, withglycerol being depleted within the first 24 hrs. Diauxic shift canclearly be observed, as no xylose is consumed until after all theglycerol has been depleted. The 20 g/L of glycerol was able to generate13 g/L of biomass. Lipid accumulation steadily occurred between 70 and230 hours, with a majority of the biomass generated on xylose beingaccounted as lipids. The culture finally achieved a biomassconcentration of 18 g/L with 7.64 g/L lipids, or 42% of total biomass.The overall productivity was 0.033 g lipids/L/hr. Strain MTYL085 wasable to convert xylose into lipids at quantities similar to other Y.lipolytica fermentations (Beopoulos et al. 2009; Papanikolaou andAggelis 2002). The yield of lipid production, however, was very low. Ofthe 80 g/L of xylose consumed, only 6.08 g/L of lipids was generated,for a yield of 0.074 g lipids/g xylose. This is only 21.7% of thetheoretical yield. This low yield may be due to overrespiration of thecarbon substrate, as high aeration on a foreign substrate may lead tostrong flux through the TCA cycle. Furthermore, 9.13 g/L citrate wasalso generated, which actually accounts for a significant yield from the100 g/L of carbon substrate initially charged. It is possible that theC/N ratio was too high, as extreme C/N ratios in Y. lipolyticafermentations can tend to produce citrate instead of lipids, likely dueto limited ability to generate sufficient ATP for fatty acid synthesis(Beopoulos et al. 2009). Despite these low yields, the vast majority(80%) of the lipids were produced after glycerol depletion and duringthe xylose-only phase, indicating successful conversion ofxylose-to-lipids using Y. lipolytica, a first step in developing acellulosic biodiesel platform.

Transcriptional Expression Affected by Secondary Substrate

To further investigate the response of Y. lipolytica duringcofermentations with xylose and the overrespiration observed onglycerol-xylose, transcriptional analysis was performed on genes withinthe TCA cycle. Xylose consumption in S. cerevisiae elicits anon-fermentative response and general upregulation of the TCA cycle (Jinet al. 2004; Salusjarvi et al. 2006). This results in lower efficienciesin xylose utilization for ethanol production as downregulation of theTCA cycle is necessary to divert carbon flux towards ethanolfermentation, whether via anaerobic environmental conditions or activityof the Crabtree effect. In our cofermentation system, the response of Y.lipolytica when transitioning from the helper substrate to xylose wasexamined. An initial RNA extraction was performed during thecofermentation while still growing on glucose, glycerol or arabitol, anda second RNA extraction was performed after the helper substrate wasdepleted and the strain was exhibiting growth on xylose as sole carbonsubstrate. RT-PCR primers used in this study are listed in Table 2. Fromthis we can identify if a similar respiratory response is observed onxylose. FIG. 5 depicts the fold-change in transcripts for pyruvatedehydrogenase (PDB1, Accession Number: XM_504448), Aconitase (ACO1,Accession Number: XM_502616), isocitrate lyase (ICL1, Accession Number:XM_501923), and isocitrate dehydrogenase (IDH1, Accession Number:XM_503571). These genes represent key enzymatic steps for theutilization of TCA cycle intermediates: PDB1, entrance into the TCAcycle; ACO1, diverting citrate to the TCA cycle instead of the cytosol;ICL1, diverting isocitrate through the glyoxylate shunt; IDH1, committedstep into oxidative respiration.

In all three cases, PDB1 is significantly upregulated, suggesting thatthere is a stronger driving force towards the TCA cycle in xylose thanany other substrate. Aconitase overexpression was not observed in theglucose-to-xylose transition, but was dramatically increased 50-fold inthe glycerol-to-xylose transition. This was mostly due to very lowtranscription levels observed of ACO1 on glycerol rather thanextraordinarily high expression of ACO1 on xylose. ACO1 was upregulatedin the transition from arabitol to xylose as well. For ICL1, significantincrease in expression was observed during the glycerol-to-xylosetransition and the arabitol-to-xylose transition, but not on glucose. Inmost organisms, ICL1 is normally not expressed due to strong cataboliterepression; however, Y. lipolytica seems to exhibit constitutiveexpression of the pathway (Flores and Gancedo 2005). Indeed, themagnitude of changes in expression of ICL1 suggests significantexpression prior to the transition. Finally, IDH1 expression is notsignificantly changed in glucose and arabitol, but is actuallydownregulated on glycerol, indicating that respiration is much morestrongly upregulated on glycerol than xylose.

The upregulation of PDB1 and ACO1 in the glycerol fermentationdemonstrate an elevated respiratory response when transitioning fromglycerol to xylose utilization. While IDH1 is downregulated, theupstream regulation may be enough to result in the overrespirationobserved in the bioreactor. It is unclear why ACO1 is downregulated sodramatically when growing on glycerol, but any previous regulation onthis enzyme must surely be alleviated. On the other hand, glucose-xylosecofermentation resulted in few significant changes in transcription.This may indicate that glucose-xylose cofermentation may yield betterresults at larger scales despite the stronger preference for glycerol byY. lipolytica.

CONCLUSION

Pentose utilization represents a pressing need in the development ofsustainable biofuel production, as the push and advantages forcellulosic feedstocks begin to outweigh the technical challenges. Theoleaginous yeast Y. lipolytica is an example of a robust platform forthe production of yeast oil that can be converted into biodiesel.Through metabolic engineering, the robust lipid production capabilitiesestablished in Y. lipolytica can be expanded to include xyloseutilization, enabling further opportunities for microbial cellulosicbiodiesel production. By testing native growth on a variety ofsubstrates we showed that the endogenous XYL3 is functional in minimalmedia, while the putative XYL12 genes are not. Through heterologousexpression of XYL1 and XYL2 genes from S. stipitis we enabled xyloseutilization in Y. lipolytica after an adaptation period. Throughcofermentation we are able to eliminate lag phases and increase growthand productivity on xylose, ultimately achieving 42% lipid accumulationin a strain that is metabolically engineered in both xylose utilizationand lipid accumulation pathways. By observing that the TCA cycleresponse, we also observed variation between cofermentation substrates,suggesting a transcriptional regulatory basis for overrespiration. Byleveraging the knowledge base developed from the study of xyloseutilization in S. cerevisiae, these results establish a framework forstudying and engineering the oleaginous yeast Y. lipolytica for xyloseutilization and the production of cellulosic biodiesel.

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EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the claims or from relevant portions of the description isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

The invention claimed is:
 1. An isolated oleaginous cell comprising anucleic acid construct that increases expression of: a xylose isomerase(XYLA) gene product, a xylose reductase (XYL1) gene product and axylitol dehydrogenase (XYL2) gene product, wherein the nucleic acidconstruct comprises an intron; and a) an expression cassette comprisinga nucleic acid sequence encoding the XYLA, XYL1, and XYL2 gene productsunder the control of a suitable homologous or heterologous promoter;and/or b) a nucleic acid sequence that modulates the level of expressionof the XYLA, XYL1, and XYL2 gene products when inserted into the genomeof the cell.
 2. The isolated oleaginous cell of claim 1, furthercomprising a nucleic acid construct that increases expression of axylulokinase (XYL3) gene product.
 3. The isolated oleaginous cell ofclaim 1, further comprising a nucleic acid construct that increasesexpression of a diacylglycerol acyltransferase (DGA) gene product, anacetyl-coA carboxylase (ACC) gene product, a stearoyl-CoA-desaturase(SCD) gene product, and/or an ATP-citrate lyase (ACL) gene product. 4.The isolated oleaginous cell of claim 1, wherein the nucleic acidconstruct inhibits or disrupts the natural regulation of a native geneencoding the gene product resulting in overexpression of the nativegene.
 5. The isolated oleaginous cell of claim 1, wherein the increasedexpression of the gene product confers a beneficial phenotype for theconversion of a carbon source to a fatty acid, fatty acid derivativeand/or triacylglycerol (TAG) to the cell.
 6. The isolated oleaginouscell of claim 5, wherein the beneficial phenotype is a modified fattyacid profile, a modified TAG profile, an increased fatty acid and/ortriacylglycerol synthesis rate, an increase conversion yield, anincreased triacylglycerol accumulation in the cell, and/or an increasedtriacylglycerol accumulation in a lipid body of the cell.
 7. Theisolated oleaginous cell of claim 6, wherein the synthesis rate, yieldor accumulation of a fatty acid or a TAG of the cell is at least 2-foldincreased as compared to unmodified cells of the same cell type.
 8. Theisolated oleaginous cell of claim 5, wherein the cell converts a carbonsource to a fatty acid or a TAG at a conversion rate within the range ofabout 0.025 g/g to about 0.32 g/g (g TAG produced/g Glucose consumed).9. The isolated oleaginous cell of claim 1, wherein the cell is anoleaginous yeast cell.
 10. The isolated oleaginous cell of claim 1,wherein the cell is a Y. lipolytica cell.
 11. A culture, comprising theisolated oleaginous cell of claim
 1. 12. The culture of claim 11,further comprising a carbon source.
 13. The culture of claim 12, whereinthe carbon source comprises a fermentable sugar.
 14. A method,comprising contacting a carbon source with an isolated oleaginous cellof claim 1, and incubating the carbon source contacted with the cellunder conditions suitable for at least partial conversion of the carbonsource into a fatty acid or a triacylglycerol by the cell.
 15. Themethod of claim 14, wherein the carbon source is a fermentable sugar.16. The method of claim 15, wherein the fermentable sugar is a C5 and/ora C6 sugar.
 17. A method for increasing productivity of production offatty acid or triacylglycerol by an isolated oleaginous cell, comprisingculturing an isolated oleaginous cell of claim 1 with at least two typesof carbon sources, wherein the first type of carbon source contains oris xylose, and wherein the second type of carbon source is a carbonsource other than xylose, whereby the productivity of production offatty acid or triacylglycerol by an isolated oleaginous cell is improvedrelative to culturing the isolated oleaginous cell or the culturewithout the second type of carbon source.
 18. The method of claim 17,wherein the second type of carbon source contains or is a C2 carbonsource, a C3 carbon source, a C5 carbon source other than xylose or a C6carbon source.